Fluorene compound

ABSTRACT

Particular compounds having a fluorene skeleton are superior in broad utility and stability, as a protecting reagent for liquid phase synthesis of amino acids and/or peptides.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationsNo. 61/159,998, filed on Mar. 13, 2009, and Japanese Patent ApplicationNo. 2009-060291, filed on Mar. 12, 2009, both of which are incorporatedherein by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to fluorene compounds which are useful asprotecting reagents for organic synthesis reactions. The presentinvention also relates to organic synthesis reactions which use such acompound and the like. More particularly, the present invention relatesto fluorene compounds which are usable as protecting reagents for aC-terminal and/or a side chain of amino acid or peptide in peptidesynthesis, particularly liquid phase synthesis of a peptide, and methodsof peptide synthesis and organic synthesis using such a compound.

2. Discussion of the Background

Methods for the organic synthesis of compounds are generally dividedlargely into solid phase methods and a liquid phase methods. The solidphase method is advantageous in that isolation and purification afterthe reaction can be performed by filtration and washing alone. However,the solid phase method is problematic in that it essentially includes anon-homogeneous phase reaction, reaction agents and reagents need to beused in excess amounts to compensate for the low reactivity, andtracking of reaction and analysis of the reaction product on a carrierare difficult.

In an attempt to perform reactions in a homogeneous liquid phase whileutilizing the advantages of the solid phase method in that isolation andpurification after the reaction can be performed by filtration andwashing alone, a method of isolating a particular component dissolved ina liquid as a solid has been used. This is because precipitation of aparticular component alone facilitates isolation and purification afterreaction.

A particular component dissolved in a solution can be precipitated onlywhen predetermined conditions, such as chemical properties, property andrelationship with solvents of the compound, are satisfied.

However, determination of precipitation conditions requires trial anderror and experimental searching in most cases. In liquid phasesynthesis, moreover, some compounds to be synthesized are insoluble inorganic solvents used for extraction or show low solubility therein,which necessitates confirmation of the property of each compound tosearch for isolation and purification methods therefor. Particularly,when sequential and multistep synthesis reactions are required as inpeptide synthesis and the like, since isolation and purificationconditions such as precipitation, extraction and the like need to bedetermined based on the properties unique to the compound synthesized ineach step, a long time and high cost are required.

To solve such problems, a method using a carrier molecule wherein adissolved state and an insolubilized state (precipitated state)irreversibly change according to the varying solvent composition hasbeen developed. Using such a carrier, an isolation target compound canbe selectively precipitated from a homogeneous solution state, in otherwords, a particular compound can be isolated after a liquid phasereaction when other soluble components still remain in a solution, thusobviating the need to consider extraction and precipitation conditionsfor each compound.

However, when a polymer is used as a carrier molecule, the reactionbecomes non-homogeneous due to the molecular weight distribution, as inthe solid phase method, where tracking of reaction and analysis of thereaction product on a carrier are difficult to perform, since thecompound is bound to a carrier.

Thus, a method using a protecting group (anchor) capable of irreversiblechange from a dissolved state to an insolubilized state (precipitatedstate) of a particular component according to the varying solventcomposition has been developed. For example, JP-A-2000-44493 and Bull.Chem. Soc. Jpn., 74, 733-738 (2001) disclose methods includingdeveloping an anchor by introducing a long chain aliphatic group into abenzyl alcohol type compound (see the following structure), dissolvingand reacting the anchor in a halogenated solvent, and precipitating areacted product with methanol or acetonitrile to allow peptide chainelongation.

However, when the anchor is used for sequential reactions of peptidesynthesis, deprotection of a second residue results in the production ofthe by-product diketopiperazine, which causes a sequence showingmarkedly decreased yield and peptide quality, thus posing problems inbroad utility (particularly sequence containing proline). With such abenzyl type anchor, moreover, deprotection under strong acidicconditions is necessary, which prevents dissociation of a desiredprotecting group.

WO2007/122847 discloses a trityl type protecting group (see thefollowing structure).

wherein m and n are each independently 0 or 1, Za is a chlorine atom ora bromine atom, and Zb is a hydroxyl group, a chlorine atom or a bromineatom.

With a protecting group of this trityl type, the formation ofdiketopiperazine as a by-product can be suppressed and selectivedeprotection can be performed. However, its property as a protectinggroup is too low, namely, the compound-anchor bond is easily cut and adecomposition reaction proceeds in which the anchor is dissociated evenin methanol and the like. Therefore, the method is not satisfactory fromthe aspects of yield and quality.

SUMMARY OF THE INVENTION

Accordingly, it is one object of the present invention to provide novelcompounds, which are superior in broad utility and stability, which areuseful as a protecting group (anchor) for amino acid and/or peptide inliquid phase synthesis and the like of peptides.

It is another object of the present invention to provide novel organicsynthesis reaction methods (particularly peptide liquid phase synthesismethod) which employ such a compound.

It is another object of the present invention to provided novel kits forliquid phase synthesis of peptides, which contain such a compound.

It is another object of the present invention to provided novelintermediates which are useful for producing such a compound.

These and other objects, which will become apparent during the followingdetailed description, have been achieved by the inventors' discoverythat a particular compound having a fluorene skeleton can solve theabove-mentioned problems.

Accordingly, the present invention provides:is as described below.

(1) A fluorene compound represented by formula (I):

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; at least one of rings A, B and C has an organic group having analiphatic hydrocarbon group; and rings A, B and C each independentlyoptionally have an electron-withdrawing group.

(2) A fluorene compound wherein 2 to 20 divalent unit structures derivedfrom a compound represented by formula (I):

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; at least one of rings A, B and C has an organic group having analiphatic hydrocarbon group; and rings A, B and C each independentlyoptionally have an electron-withdrawing group, are connected via anorganic group having an aliphatic hydrocarbon group, which is containedin the unit structure.

(3) A fluorene compound represented by formula (I′):

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; Ra, Rb and Rc are each independently an organic group having analiphatic hydrocarbon group, a hydrogen atom or an electron-withdrawinggroup, at least one of Ra, Rb and Rc is an organic group having analiphatic hydrocarbon group; and rings A, B and C each independentlyoptionally have an electron-withdrawing group.

(4) A fluorene compound represented by formula (II):

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; n is an integer of 1 to 19; Rc′ is a divalent organic grouphaving an aliphatic hydrocarbon group; rings A, B and C eachindependently optionally have one or more kinds selected from an organicgroup having an aliphatic hydrocarbon group and an electron-withdrawinggroup; when a plurality of ring A is present, respective rings A may bethe same or different; when a plurality of Y is present, respective Ymay be the same or different; and when a plurality of Rc′ is present,respective Rc′ may be the same or different.

(5) The fluorene compound of any one of the above-mentioned (1)-(4),wherein the organic group having an aliphatic hydrocarbon group and/orthe divalent organic group having an aliphatic hydrocarbon group isbonded on the ring via —O—, —S—, —NHCO— or —CONH— present in the organicgroup, or directly bonded to form a carbon-carbon bond.

(6) The fluorene compound of any one of the above-mentioned (1)-(5),wherein the organic group having an aliphatic hydrocarbon group and/orthe divalent organic group having an aliphatic hydrocarbon group has astructure comprising 2 to 10 connections of an aliphatic hydrocarbongroup bonded via —O—, —S—, —NHCO— or —CONH—, or an aliphatic hydrocarbongroup directly bonded to form a carbon-carbon bond.

(7) The fluorene compound of the above-mentioned (4), wherein thedivalent organic group having an aliphatic hydrocarbon group for Rc′ isa group represented by formula (i):

Xa-Rd

_(k1)Xa-  (i)wherein Xa is absent or —O—, —S—, —NHCO— or —CONH—; Rd is an aliphatichydrocarbon group having a carbon number of not less than 5; k₁ is aninteger of 1-10; when a plurality of Rd is present, respective Rd may bethe same or different; and when a plurality of Xa is present, respectiveXa may be the same or different.

(8) The fluorene compound of the above-mentioned (4), wherein thedivalent organic group having an aliphatic to hydrocarbon group for Rc′is a group represented by formula (ii):

O—Re

_(k2)O—  (ii)wherein Re is an aliphatic hydrocarbon group having a carbon number of 5to 60; k₂ is an integer of 1 to 3; and when a plurality of Re ispresent, respective Re may be the same or different.

(9) The fluorene compound of any one of the above-mentioned (1)-(8),wherein the organic group having an aliphatic hydrocarbon group is oneor more kinds of group selected from

a group represented by formula (a):*

X₁—R₁

_(m) ₁ H  (a)wherein * shows the position of a bond; X₁ is absent or —O—, —S—, —NHCO—or —CONH—; R₁ is an aliphatic hydrocarbon group having a carbon numberof not less than 5; m₁ is an integer of 1 to 10; when a plurality of X₁is present, respective X₁ may be the same or different; and when aplurality of R₁ is present, respective R₁ may be the same or different;

a group represented by the formula (b):

wherein * shows the position of a bond; X₂, X₂′, X₂″ and X₂′″ are eachindependently absent or —O—, —S—, —NHCO— or —CONH—; R₂ and R₄ are eachindependently a hydrogen atom, an aliphatic hydrocarbon group having acarbon number of not less than 5 or a methyl group, R₃ is an organicgroup having an aliphatic hydrocarbon group having a carbon number ofnot less than 5; n₁, n₂, n₃ and n₄ are each independently an integer of0 to 2; m₂ is an integer of 1 or 2; when a plurality of n₁, n₂, n₃ andn₄ are each present, each respective n₁, n₂, n₃ and n₄ may be the sameor different; when a plurality of X₂′, X₂″ and X₂′″ are each present,each respective X₂′, X₂″ and X₂′″ may be the same or different; and whena plurality of R₂ and R₄ are each present, each respective R₂ and R₄ maybe the same or different; and

a group represented by the formula (e):

wherein * shows the position of a bond; X₈ is absent or —O—, —S—, —NHCO—or —CONH—; m₃ is an integer of 0 to 15; n₅ is an integer of 0 to 11; n₆is an integer of 0 to 5; X₇ is absent or —O—, —S—, —COO—, —OCONH—,—NHCO— or —CONH—; R₁₂ is a hydrogen atom, a methyl group or an aliphatichydrocarbon group having a carbon number of not less than 5; when aplurality of X₇ is present, respective X₇ may be the same or different;and when a plurality of R₁₂ is present, respective R₁₂ may be the sameor different.

(10) The fluorene compound of the above-mentioned (9), wherein,

in the formula (a), X₁ is —O—; R₁ is an aliphatic hydrocarbon grouphaving a carbon number of 5 to 60; m₁ is 1;

in the formula (b), X₂ is —O—, or —CONH—; X₂′, X₂″ and X₂′″ are eachindependently absent, or —O—; R₂ and R₄ are each independently analiphatic hydrocarbon group having a carbon number of 5 to 60 or amethyl group; R₃ is an organic group having an aliphatic hydrocarbongroup having a carbon number 5 to 60; n₁, n₂, n₃ and n₄ are the same ordifferent and each is an integer of 0 or 1; m₂ is 1, and

in the formula (e), X₈ is —O—; m₃ is 2 or 3; n₅ is 1; n₆ is 2 or 3; X₇is —O—; and R₁₂ in the number of m₃ are each independently an alkylgroup having a carbon number of 8 to 60.

(11) The fluorene compound of any one of the above-mentioned (1)-(6),wherein the aliphatic hydrocarbon group of the organic group having analiphatic hydrocarbon group and/or the divalent organic group having analiphatic hydrocarbon group is an aliphatic hydrocarbon group having acarbon number of not less than 5.

(12) The fluorene compound of any one of the above-mentioned (1)-(6),wherein the aliphatic hydrocarbon group of the organic group having analiphatic hydrocarbon group and/or the divalent organic group having analiphatic hydrocarbon group is an aliphatic hydrocarbon group having acarbon number of 5 to 60.

(13) The fluorene compound of any one of the above-mentioned (1)-(12),wherein the total carbon number derived from an aliphatic hydrocarbongroup in one molecule is not less than 20.

(14) The fluorene compound of any one of the above-mentioned (1)-(12),wherein the total carbon number derived from an aliphatic hydrocarbongroup in one molecule is 20 to 200.

(15) The fluorene compound of any one of the above-mentioned (1)-(14),wherein Y is a hydroxyl group, a bromo group, a chloro group, an iodogroup, a thiol group or an amino group.

(16) The fluorene compound of any one of the above-mentioned (1)-(14),wherein Y is a hydroxyl group, a bromo group or a chloro group.

(17) The fluorene compound of any one of the above-mentioned (1)-(16),wherein the aromatic ring of ring A is a benzene ring.

(18) The fluorene compound of the above-mentioned (3), wherein Ra is ahalogen atom.

(19) The fluorene compound of the above-mentioned (3), wherein theorganic group having an aliphatic hydrocarbon group is present at the2-position and/or the 7-position of the fluorene compound.

(20) The fluorene compound of the above-mentioned (3), wherein Rb and/orRc are/is

a group represented by the formula (a) wherein m₁ is l; X₁ is —O—; andR₁ is an aliphatic hydrocarbon group having a carbon number of 5 to 60,

a group represented by the formula (b) wherein X₂, X₂′, X₂″ and X₂′″ areeach —O—; R₂ and R₄ are each independently an aliphatic hydrocarbongroup having a carbon number of 5 to 60; R₃ is an organic group havingan aliphatic hydrocarbon group having a carbon number of 5 to 60; n₁,n₂, n₃ and n₄ are each 1; and m₂ is 1, or

a group represented by the formula (e) wherein X₈ is —O—; m₃ is 2 or 3;n₅ is 1; n₆ is 3; X₇ is —O—; and R₁₂ in the number of m₃ are eachindependently an alkyl group having a carbon number of 14 to 30.

(21) The fluorene compound of the above-mentioned (3), wherein ring A isa benzene ring; Y is a hydroxyl group, a bromo group or a chloro group;Ra is a halogen atom; and an organic group having an aliphatichydrocarbon group is a group represented by the formula (a) wherein m₁is 1; X₁ is —O—; and R₁ is an aliphatic hydrocarbon group having acarbon number of 5 to 60, a group represented by the formula (b) whereinX₂, X₂′, X₂″ and X₂′″ are each —O—; R₂ and R₄ are each independently analiphatic hydrocarbon group having a carbon number of 5 to 60; R₃ is anorganic group having an aliphatic hydrocarbon group having a carbonnumber 5 to 60; n₁, n₂, n₃ and n₄ are each 1; and m₂ is 1, or a grouprepresented by the formula (e) wherein X₈ is —O—; m₃ is 2 or 3; n₅ is 1;n₆ is 3; X₇ is —O—; and R₁₂ in the number of m₃ are each independentlyan alkyl group having a carbon number of 14 to 30, each of whichformulas is present at the 2-position and/or the 7-position of thefluorene compound.

(22) The fluorene compound of the above-mentioned (4), wherein n is 1.

(23) The fluorene compound of the above-mentioned (4), wherein ring Ahas an electron-withdrawing group.

(24) The fluorene compound of the above-mentioned (23), wherein theelectron-withdrawing group is a halogen atom.

(25) The fluorene compound of the above-mentioned (4), wherein Rc′ is agroup represented by the formula (i) wherein Xa is —O—; Rd in the numberof k₁ are each independently an aliphatic hydrocarbon group having acarbon number of 5 to 60, and k₁ is an integer of 1 to 3.

(26) The fluorene compound of the above-mentioned (4), wherein ring A isa benzene ring; Y is a hydroxyl group, a bromo group or a chloro group;n is 1; ring A has a halogen atom as the electron-withdrawing group; Rc′is a group represented by the formula (i) wherein Xa is —O—; Rd is analiphatic hydrocarbon group having a carbon number of 5 to 60, and k₁ isan integer of 1 to 3.

(27) The fluorene compound of the above-mentioned (3) or (4), which isselected from the group consisting of

-   2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol;-   2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene;-   2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromofluorene;-   2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene;-   1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane;-   1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane;-   2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol;-   2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene;-   9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenol;    and-   9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-bromofluorene.

(28) A protecting reagent for organic synthesis reaction, comprising thefluorene compound of any one of the above-mentioned (1)-(27).

(29) A protecting reagent for a carboxyl group of amino acid or peptide,comprising the fluorene compound of any one of the above-mentioned(1)-(27).

(30) A protecting reagent for the C-terminal of amino acid or peptide,comprising the fluorene compound of any one of the above-mentioned(1)-(27).

(31) A method of producing a peptide by a liquid phase synthesis processcomprising the following steps;

(a) a step of binding a fluorene compound of any one of theabove-mentioned (1)-(27) to an amino acid or peptide (binding step), and

(b) a step of precipitating the bonded product of the compound and theamino acid or peptide obtained in the above-mentioned step(precipitation step).

(32) A method of producing a peptide by a liquid phase synthesis processcomprising the following steps;

(a) a step of obtaining C-fluorene compound-protected amino acid orC-fluorene compound-protected peptide by condensing a fluorene compoundof any one of the above-mentioned (1)-(27) with the C-terminal of anN-protected amino acid or N-protected peptide (C-terminal fluorenecompound protection step),

(b) a step of removing the protecting group of the N-terminal of theamino acid or peptide obtained in the above-mentioned step (N-terminaldeprotection step),

(c) a step of condensing the N-terminal of the amino acid or peptideobtained in the above-mentioned step with N-protected amino acid orN-protected peptide (peptide chain elongation step), and

(d) a step of precipitating the peptide obtained in the above-mentionedstep (precipitation step).

(33) The method of the above-mentioned (32), further comprising one ormore repeats of the following steps (e)-(g):

(e) a step of deprotecting the N-terminal of the peptide obtained in theprecipitation step,

(f) a step of condensing the N-terminal of peptide obtained in theabove-mentioned step with N-protected amino acid or N-protected peptide,and

(g) a step of precipitating the peptide obtained in the above-mentionedstep.

(34) A method of producing a peptide further comprising, after the finalprecipitation step of the above-mentioned (32) or (33), a step ofdeprotecting the C-terminal of peptide wherein the C-terminal isprotected with a fluorene compound.

(35) A method of producing a peptide compound, comprising using afluorene compound of any one of the above-mentioned (1)-(27).

(36) A method of producing an organic compound, 35 comprising using afluorene compound of any one of the above-mentioned (1)-(27).

(37) A compound represented by the following formula (III):

wherein X₂′, X₂″ and X₂′″ are each independently absent or —O—, —S—,—NHCO— or —CONH—; R₂ and R₄ are each independently a hydrogen atom, analiphatic hydrocarbon group having a carbon number of not less than 5 ora methyl group; R₃ is an organic group having an aliphatic hydrocarbongroup having a carbon number of not less than 5; n₁, n₂, n₃ and n₄ areeach independently an integer of 0 to 2; m₂ is an integer of 1 or 2;when a plurality of n₁, n₂, n₃ and n₄ are each present, each respectiven₁, n₂, n₃ and n₄ may be the same or different; when a plurality of X₂′,X₂″ and X₂′″ are each present, each respective X₂′, X₂″ and X₂′″ may bethe same or different; and a plurality of when R₂ and R₄ are eachpresent, each respective R₂ and R₄ may be the same or different; and Halis a halogen atom.

(38) The compound of the above-mentioned (37), wherein, in the formula(III), X₂′, X₂″ and X₂′″ are the same or different and each is absent or—O—; R₂ and R₄ are each independently an aliphatic hydrocarbon grouphaving a carbon number of 10 to 30 or a methyl group; R₃ is an organicgroup having an aliphatic hydrocarbon group having a carbon number of 10to 30; n₁, n₂, n₃ and n₄ are the same or different and each is aninteger of 0 or 1; and m₂ is 1.

(39) The compound of the above-mentioned (37), which is1-(3-iodo-2,2-bis-octadecanoxymethyl-propoxy)octadecane.

The compounds of the present invention are superior in broad utility andstability in organic synthesis reactions, particularly peptide synthesisreactions. When peptide synthesis is performed using a compound of thepresent invention as an anchor, a sequence that easily formsdiketopiperazine can be synthesized in a high yield, and further, theanchor can be selectively removed. Thus, a protected peptide can bedirectly obtained without removing other protecting groups (peptidesidechain protecting group etc.).

The particular compounds of the present invention having a fluoreneskeleton function as a superior protecting group and anchor, and theanchor can be removed under weak acidic conditions, and selectivelyremoved even when other protecting groups (peptide side chain protectinggroup etc.) still remain in the compound obtained by organic synthesisreactions. Namely, the compound is a substance that enables easyprecipitation in methanol and the like, since it dissolves only inhalogen solvents, THF and the like and scarcely dissolves in polarorganic solvents. In the process of elongation of peptide chain lengthwherein the substance is used as a protecting group for C-terminal orside chain in the peptide synthesis and an operation including reactionin a halogen solvent, followed by precipitation with methanol and thelike to remove impurity is repeated, a side reaction givingdiketopiperazine is suppressed and the peptide chain can be elongated ina high yield and with high quality. Moreover, the anchor enablesselective removal thereof while maintaining the protecting group.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise specified in the sentences, any technique terms andscientific terms used in the present specification, have the samemeaning as those generally understood by those of ordinary skill in theart in the art the present invention belongs to. Any methods andmaterials similar or equivalent to those described in the presentspecification can be used for practicing or testing the presentinvention, and preferable methods and materials are described in thefollowing. All publications and patents referred to in the Specificationare hereby incorporated by reference so as to describe and discloseconstructed products and methodology described in, for example,publications usable in relation to the described invention.

1. The Compounds of the Present Invention

The compound of the present invention is useful as a reagent for organicsynthesis. Here, the reagent for organic synthesis refers to any reagentrelating to organic synthesis reactions, and is a concept includingreagents directly involved in the reaction such as reaction substrate,reaction promoter, reagent for introducing protecting group,deprotecting agent and the like, as well as inert solvent and the like.Specifically, reagents to be used for peptide synthesis reaction areexemplified. Preferred is a reagent introduced as a protecting group(anchor) of the C-terminal of amino acid or peptide in liquid phasesynthesis of peptide, and a reagent introduced as a protecting group asa peptideside chain protecting group in peptide synthesis reaction, andan appropriate compound can be selected according to the object.Particularly preferable compounds of the present invention are anchors.

One embodiment of the compound of the present invention is a fluorenecompound represented by the following formula (I):

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; at least one of rings A, B and C has an organic group having analiphatic hydrocarbon group; and rings A, B and C each independentlyoptionally have an electron-withdrawing group.

A fluorene compound represented by the following formula (I′), whereinat least one of rings A, B and C has “an organic group having analiphatic hydrocarbon group” and the number of “an organic group havingan aliphatic hydrocarbon group” of each ring is 1 to 4, preferably 1 to3, more preferably 1 or 2, still more preferably 1 or 0, is morepreferable.

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; Ra, Rb and Rc are each independently an organic group having analiphatic hydrocarbon group, a hydrogen atom or an electron-withdrawinggroup, and at least one of Ra, Rb and Rc is an organic group having analiphatic hydrocarbon group; and rings A, B and C each independentlyoptionally have an electron-withdrawing group.

A fluorene compound represented by the formula (I′) is encompassed in afluorene compound represented by the formula (I).

Moreover, the compound of the present invention may be a compound havinga structure as shown below, wherein a plurality of divalent unitstructures derived from a compound represented by the formula (I) areconnected. Preferred is a fluorene compound wherein 2 to 20 of adivalent unit structure derived from a compound represented by thefollowing formula (I):

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; at least one of rings A, B and C has an organic group having analiphatic hydrocarbon group; and rings A, B and C each independentlyoptionally have an electron-withdrawing group,are connected via an organic group having an aliphatic hydrocarbongroup, which the unit structure has.

As the above-mentioned compound, a compound represented by the followingformula (II) is also preferable.

wherein ring A is an aromatic ring; Y is a group reactive with one ormore kinds selected from an amino group, a carboxyl group and a mercaptogroup; n is an integer of 1 to 19; Rc′ is a divalent organic grouphaving an aliphatic hydrocarbon group; rings A, B and C eachindependently optionally have one or more kinds selected from an organicgroup having an aliphatic hydrocarbon group and an electron-withdrawinggroup; when a plurality of ring A is present, respective rings A may bethe same or different; when a plurality of Y is present, respective Ymay be the same or different; and when plurality of Rc′ is present,respective Rc′ may be the same or different.

The divalent organic group having an aliphatic hydrocarbon group for Rc′is preferably represented by the following formula (i).

Xa-Rd

_(k1)Xa-  (i)wherein Xa is absent or —O—, —S—, —NHCO—, or —CONH—; Rd is an aliphatichydrocarbon group having a carbon number of not less than 5; k₁ is aninteger of 1 to 10; when a plurality of Rd is present, respective Rd maybe the same or different; and when a plurality of Xa is present,respective Xa may be the same or different.

In formula (i), Xa is preferably —O—, Rd is preferably an aliphatichydrocarbon group having a carbon number of 5 to 60, and k₁ ispreferably an integer of 1 to 3. That is, preferably formula (i) isrepresented by the following formula (ii).

O—Re

_(k2)O—  (ii)wherein Re is an aliphatic hydrocarbon group having a carbon number of 5to 60; k₂ is an integer of 1 to 3; and when a plurality of Re ispresent, respective Re may be the same or different.

In the present specification, the “aromatic ring” means an “aromatichydrocarbon ring” or an “aromatic heterocycle”.

As the “aromatic hydrocarbon ring”, C₆₋₁₄ arene (e.g., benzene,naphthalene and the like) is preferable, C₆₋₁₀ arene is more preferable,and benzene is particularly preferable.

Examples of the “aromatic heterocycle” include a 5- to 7-memberedmonocyclic aromatic heterocycle or condensed aromatic heterocyclecontaining, as a ring-constituting atom besides carbon atom, 1 to 4hetero atoms selected from an oxygen atom, a sulfur atom and a nitrogenatom. Examples of the condensed aromatic heterocycle include a groupwherein such 5- to 7-membered monocyclic aromatic heterocycle and a6-membered ring containing 1 or 2 nitrogen atoms, benzene ring, or a5-membered ring containing one sulfur atom are condensed and the like.

Preferable examples of the “aromatic heterocycle” include 5 to7-membered monocyclic aromatic heterocycle such as furan, thiophene,pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole, pyrazole,1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole, furazan,1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,3,4-thiadiazole, 1,2,3-triazole,1,2,4-triazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine,triazine and the like; 8- to 16-membered (preferably 8- to 12-membered)condensed aromatic heterocycle (preferably heterocycle wherein one ortwo (preferably one) 5- to 7-membered monocyclic aromatic heterocyclementioned above is condensed with one or two (preferably one) benzenerings, or heterocycle wherein the same or different two or three(preferably two) 5- to 7-membered monocyclic aromatic heterocyclesmentioned above are condensed) such as benzofuran, isobenzofuran,benzo[b]thiophene, benzo[c]thiophene, indole, isoindole, 1H-indazole,benzimidazole, benzoxazole, 1,2-benzisoxazole, benzothiazole,1,2-benzisothiazole, 1H-benzotriazole, quinoline, isoquinoline,cinnoline, quinazoline, quinoxaline, phthalazine, naphthyridine, purine,pteridinee, carbazole, α-carboline, β-carboline, γ-carboline, acridine,phenoxathiine, phenothiazine, phenazine, phenoxathiine, thianthrene,phenanthridine, phenanthrolin, indolizine, pyrrolopyridine,pyrrolo[1,2-b]pyridazine, pyrazolo[1,5-a]pyridine,imidazo[1,2-a]pyridine, imidazo[1,5-a]pyridine,imidazo[1,2-b]pyridazine, imidazo[1,2-a]pyrimidine,1,2,4-triazolo[4,3-a]pyridine, 1,2,4-triazolo[4,3-b]pyridazine, and thelike, and the like.

When desired, the “aromatic ring” optionally further has substituent(s)besides the organic group having an aliphatic hydrocarbon group in theformula (I) and the Ra group in the formula (I′). Examples of thefurther substituent include an electron-withdrawing group and the like.Specific examples of the further substituent include a halogen atom(chlorine atom, bromine atom, fluorine atom, iodine atom), optionallyhalogenated alkyl group (trifluoromethyl group etc.), nitro group, cyanogroup, ester group [alkoxycarbonyl group (methoxycarbonyl group,ethoxycarbonyl group, propoxycarbonyl group etc.), aryloxycarbonyl group(phenyloxycarbonyl group etc.)] and the like.

In the present specification, examples of the “group reactive with anamino group, a carboxyl group and/or a mercapto group” include ahydroxyl group, a bromo group, a chloro group, an iodo group, a thiolgroup, an amino group and the like, with preference given to a hydroxylgroup, a bromo group and a chloro group.

The amino group, carboxyl group and/or mercapto group are present in anamino acid or peptide, which is a starting material of the peptidesynthesis. That is, the compound represented by the formula (I), theformula (I′) or the formula (II) of the present invention is bonded toamino acid or peptide via Y group which is a “group reactive with anamino group, a carboxyl group and/or a mercapto group”. Preferably, inthe liquid phase synthesis of peptide, it can function as an anchor ofthe C-terminal (C-terminal protecting reagent) of an amino acid orpeptide to be the starting material, and/or as a side chain-protectinggroup (e.g., a carboxyl-protecting reagent).

Here, the “side chain” is a chained structure other than the main chainpresent in an amino acid or peptide to be the starting material. Forexample, an acidic amino acid such as aspartic acid and glutamic acidalso has a carboxyl group in the side chain, an amino group in lysineand mercapto group in cysteine.

In the present specification, the “electron-withdrawing group” includesthose generally used in the art. Specifically, a halogen atom (chlorineatom, bromine atom, fluorine atom, iodine atom), a nitro group, aperhalogenoalkyl group (e.g., trifluoromethyl group), a cyano group, acarboxyl group, an alkoxycarbonyl group (e.g., methoxycarbonyl group,ethoxycarbonyl group), an aryloxycarbonyl group (e.g., phenyloxycarbonylgroup), an alkylamide group (e.g., acetamide group), aperhalogenoalkylamide group (e.g., trifluoroacetamido group), anoptionally substituted alkanoyl group (e.g., acetyl group, ethanoylgroup, propanoyl group, isopropanoyl group, butanoyl group, isobutanoylgroup, trifluoroacetyl group), an optionally substituted aroyl group(e.g., benzoyl group, naphthylcarbonyl group, p-chlorobenzoyl group), anoptionally substituted alkylsulfonyl group (e.g., methanesulfonyl group,trifluoromethanesulfonyl group), an optionally substituted arylsulfonylgroup (e.g., benzenesulfonyl group, p-toluenesulfonyl group), anoptionally substituted alkylsulfoneamide group (e.g.,methanesulfoneamide group, trifluoromethanesulfoneamide group), anoptionally substituted arylsulfonamide group (e.g., benzenesulfoneamidegroup or p-toluenesulfoneamide group) and the like can be mentioned.Preferred are fluorine atom, chlorine atom, nitro group, trifluoromethylgroup, cyano group, trifluoroacetamido group, trifluoroacetyl group,p-chlorobenzoyl group, methanesulfonyl group, trifluoromethanesulfonylgroup, benzenesulfonyl group, p-toluenesulfonyl group,methanesulfoneamide group, trifluoromethanesulfoneamide group,benzenesulfoneamide group, p-toluenesulfoneamide and the like, andparticularly preferred are halogen atom and trifluoromethyl group.

In the present specification, the “organic group having an aliphatichydrocarbon group” means a monovalent organic group (one bond is bondedto at least one of rings A, B and C) which has an aliphatic hydrocarbongroup in the molecular structure.

The “aliphatic hydrocarbon group” of the “organic group having analiphatic hydrocarbon group” is a aliphatic hydrocarbon group consistingof straight chain or branched, saturated or unsaturated carbon andhydrogen, and an aliphatic hydrocarbon group having a carbon number ofnot less than 5 is preferable, an aliphatic hydrocarbon group having acarbon number of 5 to 60 is particularly preferable, an aliphatichydrocarbon group having a carbon number of 5 to 30 is more preferable,and an aliphatic hydrocarbon group having a carbon number of 10 to 30 isfurther preferable.

The site of the “aliphatic hydrocarbon group” in the organic grouphaving an aliphatic hydrocarbon group of is not particularly limited,and it may be present at the terminal (monovalent group), or other site(for example, divalent group).

Specific examples of the aliphatic hydrocarbon group include monovalentgroups such as methyl group, ethyl group, propyl group, isopropyl group,butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentylgroup, hexyl group, octyl group, decyl group, lauryl group, tridecylgroup, myristyl group, cetyl group, stearyl group, aralkyl group,behenyl group, oleyl group, isostearyl group and the like and a divalentgroup derived therefrom.

In the “organic group having an aliphatic hydrocarbon group”, a moietyother than the aliphatic hydrocarbon group can be set freely. Forexample, it optionally has a moiety such as —O—, —S—, —NHCO—, —CONH—,hydrocarbon group and the like. Examples of the “hydrocarbon group”include a monovalent group such as aliphatic hydrocarbon group,monocyclic saturated hydrocarbon group and aromatic hydrocarbon groupand the like, specific examples include alkyl group, alkenyl group,alkynyl group, cycloalkyl group, aryl group and the like and a divalentgroup derived therefrom. Examples of the “alkyl group” include C₁₋₆alkyl group and the like and preferable examples include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyland the like. Examples of the “alkenyl group” include C₂₋₆ alkenyl groupand the like and preferable examples include vinyl, 1-propenyl, allyl,isopropenyl, butenyl, isobutenyl and the like. Examples of “alkynylgroup” include C₂₋₆ alkynyl group and the like and preferable examplesinclude ethynyl, propargyl, 1-propynyl and the like. Examples of the“cycloalkyl group” include C₃₋₆ cycloalkyl group and the like andpreferable examples include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl. The “aryl group” is preferably, for example, C₆₋₁₄ arylgroup such as phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, 2-anthryl andthe like, and the like, more preferably C₆₋₁₀ aryl group and, forexample, phenyl group and the like. The hydrocarbon group is optionallysubstituted by a substituent selected from a halogen atom (chlorineatom, bromine atom, fluorine atom, iodine atom), an oxo group and thelike.

The “organic group having an aliphatic hydrocarbon group” may be bonded(substitution) to at least one of rings A, B and C via an aliphatichydrocarbon group present in the group, namely, directly bonded to forma carbon-carbon bond or via —O—, —S—, —NHCO—, —CONH— and the likepresent in the group. Preferably, it is bonded via —O—, —S— or —CONH— inview of the easiness of synthesis of the compound. More preferably, itis bonded via —O—.

The compound of in the present invention is characterized in that ringA, B and/or C are/is substituted by 1 to 4, preferably 1 to 3, morepreferably 1 or 2, “organic groups having an aliphatic hydrocarbongroup” mentioned above. A same ring may be substituted by all “organicgroups having an aliphatic hydrocarbon group”, a different ring may besubstituted thereby. In addition, plural aliphatic hydrocarbon groupsmay be contained in one “organic group having an aliphatic hydrocarbongroup” by branching and the like. In the compound of the presentinvention, when a plurality of aliphatic hydrocarbon group is present,they may be the same or different.

In the compound of the present invention, an organic group having analiphatic hydrocarbon group is unlimitatively preferably bonded to the2-position and/or the 7-position on the fluorene ring in the compound ofthe present invention, since the final deanchoring is easy.

The “aliphatic hydrocarbon group” in the compound of the presentinvention is appropriately selected according to the use of the compoundto be synthesized. For example, when it is use as a peptidesidechain-protecting group in the peptide synthesis, one having acomparatively short chain length, for example, one having a carbonnumber of less than 5 can be employed. When it is used as an anchor atthe C-terminal of an amino acid or peptide, one having a comparativelylong chain length, namely, one having a carbon number of not less than 5is preferably employed. Furthermore, an aliphatic hydrocarbon grouphaving a carbon number of 5 to 60 is more preferable, an aliphatichydrocarbon group having a carbon number of 5 to 30 is furtherpreferable, and an aliphatic hydrocarbon group having a carbon number of10 to 30 is still more preferable. When it is used as an anchor, thetotal number of carbon derived from the “aliphatic hydrocarbon group” inone molecule of the compound of the present invention (hereinafter to bealso referred to as the total carbon number derived from aliphatichydrocarbon group; when a plurality of “aliphatic hydrocarbon group” ispresent, the total thereof) is preferably not less than 20, morepreferably 20 to 200, further preferably 20 to 100, and still morepreferably 20 to 60. When the carbon number is higher, crystallinity ofthe compound of the present invention in a polar organic solvent becomesfine even when the peptide chain is a long chain.

Examples of the “organic group having an aliphatic hydrocarbon group”include the groups represented by the following formulas (a) to (e).*

X₁—R₁

_(m) ₁ H  (a)

In the formula (a), * shows the position of a bond; X₁ is absent or —O—,—S—, —NHCO— or —CONH—; R₁ is an aliphatic hydrocarbon group having acarbon number of not less than 5; m₁ is an integer of 1 to 10; when aplurality of X₁ is present, respective X₁ may be the same or different;and when a plurality of R₁ is present, respective R₁ may be the same ordifferent.

In the formula (a), particularly a group wherein X₁ is —O—; R₁ is analiphatic hydrocarbon group having a carbon number of 5 to 60; and m₁ is1, is preferable.

As the “aliphatic hydrocarbon group having a carbon number of not lessthan 5” for R₁, an “aliphatic hydrocarbon group” of the above-mentioned“organic group having an aliphatic hydrocarbon group”, which has acarbon number of not less than 5 can be mentioned, with preference givento on having a carbon number of 5 to 60.

In the formula (b), shows the position of a bond; X₂, X₂′, X₂″ and X₂′″are each independently absent, or —O—, —S—, —NHCO— or —CONH—; R₂ and R₄are each independently a hydrogen atom, an aliphatic hydrocarbon grouphaving a carbon number of not less than 5 or a methyl group; R₃ is anorganic group having an aliphatic hydrocarbon group having a carbonnumber of not less than 5; n₁, n₂, n₃ and n₄ are each independently aninteger of 0 to 2; m₂ is an integer of 1 or 2; when a plurality of n₁,n₂, n₃ and n₄ are present, each respective n₁, n₂, n₃ and n₄ may be thesame or different; when a plurality of X₂′, X₂″ and X₂′″ are present,each respective X₂′, X₂″ and X₂′″ may be the same or different; and whena plurality of R₂ and R₄ are present, each respective R₂ and R₄ may bethe same or different.

As the “aliphatic hydrocarbon group having a carbon number of not lessthan 5” for R₂, R₃ or R₄, from among the above-mentioned “organic grouphaving an aliphatic hydrocarbon group”, an “aliphatic hydrocarbon group”having a carbon number of not less than 5 can be mentioned, withpreference given to one having a carbon number of 5 to 60.

In the formula (b), a group wherein X₂ is —O—, —NHCO— or —CONH—,preferably —O— or —CONH—; X₂′, X₂″ and X₂′″ are each independentlyabsent or —O—; R₂ and R₄ are each independently an aliphatic hydrocarbongroup having a carbon number of 5 to 60 or a methyl group; R₃ is anorganic group having an aliphatic hydrocarbon group having a carbonnumber 5 to 60; n₁, n₂, n₃ and n₄ are the same or different and each isan integer of 0 or 1; and m₂ is 1, is particularly preferable.

In the formula (b), a group wherein X₂, X₂′, X₂″ and X₂′″ are each —O—,R₂ and R₄ are each independently an aliphatic hydrocarbon group having acarbon number of 5 to 60, R₃ is an organic group having an aliphatichydrocarbon group having a carbon number 5 to 60, n₁, n₂, n₃ and n₄ areeach 1, and m₂ is 1, is more preferable.*—X₃—R₅—Ar—X_(3′—R) ₆  (c)

In the formula (c), shows the position of a bond; X₃ and X₃′ are thesame or different and each is absent or —O—, —S—, —NHCO— or —CONH—; R₅and R₆ are the same or different and each is an aliphatic hydrocarbongroup; and Ar is an arylene group.

Examples of the “arylene group” include phenylene, naphthylene,biphenylene and the like, with preference given to phenylene.

As the “aliphatic hydrocarbon group” for R₅ or R₆, those similar to the“aliphatic hydrocarbon group” of the above-mentioned “organic grouphaving an aliphatic hydrocarbon group” can be mentioned, particularlyone having a carbon number of not less than 5 preferably 5 to 60, can bementioned.

In the formula (c), a group wherein X₃ and X₃′ are each —O—; R₅ and R₆are each independently an aliphatic hydrocarbon group having a carbonnumber of 5 to 60; and Ar is phenylene, is particularly preferable.

In the formula (d), * shows the position of a bond; o and p are each 0or 1; X₄, X₄′, X₄″, X₅ and X₆ are the same or different and each isabsent, or —O—, —S—, —NHCO— or —CONH—; R₇, R₈ and R₉ are the same ordifferent and each is an aliphatic hydrocarbon group and R₁₀ and R₁₁ areeach an alkylene group having a carbon number of 1 to 10.

Examples of the “alkylene group” having a carbon number of 1 to 10include methylene, ethylene, propylene, butylene, hexylene, octylene,dodecylene and the like.

As the “aliphatic hydrocarbon group” for R₇-R₉, those similar to the“aliphatic hydrocarbon group” of the above-mentioned “organic grouphaving an aliphatic hydrocarbon group” can be mentioned, particularlyone having a carbon number of not less than 5 can be mentioned,preferably one having a carbon number of 5 to 60.

In the formula (d), a group wherein o is 0; p is 1; X₄, X₄′ and X₄″ areeach —O—; X₅ is —NHCO—; X₆ is —O—; R₇-R₉ is an aliphatic hydrocarbongroup having a carbon number of 5 to 60; R₁₀ and R₁₁ are each analkylene group having a carbon number of 1 to 10, is particularlypreferable.

wherein * shows the position of a bond; X₈ is absent or —O—, —S—, —NHCO—or —CONH—; m₃ is an integer of 0 to 15; n₅ is an integer of 0 to 11; n₆is an integer of 0 to 5; X₇ is absent or —O—, —S—, —COO—, —OCONH—,—NHCO— or —CONH—; R₁₂ is a hydrogen atom, a methyl group or an aliphatichydrocarbon group having a carbon number of not less than 5; when aplurality of X₇ is present, respective X₇ may be the same or different;and when a plurality of R₁₂ is present, respective R₁₂ may be the sameor different.

As the “aliphatic hydrocarbon group having a carbon number of not lessthan 5” for R₁₂, the “aliphatic hydrocarbon group” of theabove-mentioned “organic group having an aliphatic hydrocarbon group”having a carbon number of not less than 5 can be mentioned, preferablyone having a carbon number of 5 to 80.

In the formula (e), a group wherein X₈ is —O—; m₃ is 2 or 3; n₅ is 1; n₆is 2 or 3; X₇ is —O—; and R₁₂ in the number of m₃ are each independentlyan alkyl group having a carbon number of 8 to 60, is preferable.

In the formula (e), a group wherein X₈ is —O—, m₃ is 2 or 3, n₅ is 1, n₆is 3, X₇ in the number of m₃ is —O—, and R₁₂ in the number of m₃ areeach independently an alkyl group having a carbon number of 14 to 30, isparticularly preferable.

Specific examples of the “organic group having an aliphatic hydrocarbongroup” from among aliphatic carbon chain groups having a carbon numberof 18 or 22 include the following. In each group, * shows the positionof a bond.

In addition, the following group is also used.

In the present specification, “the divalent organic group having analiphatic hydrocarbon group” is a divalent organic group having analiphatic hydrocarbon group in a molecule structure thereof.

As the “aliphatic hydrocarbon group” of the “divalent organic grouphaving an aliphatic hydrocarbon group”, those similar to the “aliphatichydrocarbon group” of the above-mentioned “organic group having analiphatic hydrocarbon group” can be mentioned.

A fluorene compound wherein 2 to 20 divalent unit structures derivedfrom a compound represented by the formula (I) are connected via the“divalent organic group having an aliphatic hydrocarbon group” is alsowithin the range of the present invention.

Examples of such divalent organic group include those represented by thefollowing formula (i):

Xa-Rd

_(k1)Xa-  (i)wherein Xa is absent, or —O—, —S—, —NHCO— or —CONH—; Rd is an aliphatichydrocarbon group having a carbon number of not less than 5; k₁ is aninteger of 1 to 10; when a plurality of Rd is present, respective Rd maybe the same or different; and when a plurality of Xa is present,respective Xa may be the same or different, and represented by thefollowing formula (ii):

O—Re

_(k2)O—  (ii)wherein Re is an aliphatic hydrocarbon group having a carbon number of 5to 60; k₂ is an integer of 1 to 3; and when Re is present in plurality,respective Re may be the same or different, are more preferable.

As the “aliphatic hydrocarbon group having a carbon number of not lessthan 5” for Rd, the “aliphatic hydrocarbon group” of the above-mentioned“organic group having an aliphatic hydrocarbon group” having a carbonnumber of not less than 5 can be mentioned.

As the “aliphatic hydrocarbon group having a carbon number of 5 to 60”for Re, the “aliphatic hydrocarbon group” of the above-mentioned“organic group having an aliphatic hydrocarbon group” having a carbonnumber of 5 to 60 can be mentioned.

As a fluorene compound represented by the formula (I) of the presentinvention, a compound represented by the formula (I′) is preferable,more preferably a compound represented by the formula (I′),

wherein ring A is a benzene ring; Y is a hydroxyl group, a bromo groupor a chloro group; Ra is a halogen atom; an organic group having analiphatic hydrocarbon group is a group represented by the formula (a)wherein m₁ is 1; X₁ is —O—; R₁ is an aliphatic hydrocarbon group havinga carbon number of 5 to 60, a group represented by the formula (b)wherein X₂, X₂′, X₂″ and X₂′″ are each —O—; R₂ and R₄ are eachindependently an aliphatic hydrocarbon group having a carbon number of 5to 60; R₃ is an organic group having an aliphatic hydrocarbon grouphaving a carbon number 5 to 60; n₁, n₂, n₃ and n₄ are each 1; and m₂ is1, or a group represented by the formula (e) wherein X₈ is —O—; m₃ is 2or 3; n₅ is 1; n₆ is 3; X₇ is —O—; R₁₂ in the number of m₃ are eachindependently an alkyl group having a carbon number of 14 to 30, each ofwhich formulas is present at the 2-position and/or the 7-position of thefluorene compound.

A fluorene compound represented by the formula (II) of the presentinvention is preferably a compound represented by the formula (II),

wherein ring A is a benzene ring; Y is a hydroxyl group, a bromo groupor a chloro group; n is 1; ring A has a halogen atom as theelectron-withdrawing group; Rc′ is a group represented by the formula(i) wherein Xa is —O—; Rd is an aliphatic hydrocarbon group having acarbon number of 5 to 60, and k₁ is an integer of 1 to 3.

Preferable examples of the fluorene compound of the present inventioninclude the following fluorene compounds.

-   2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol;-   2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene;-   2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromofluorene;-   2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene;-   1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane;-   1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane;-   2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol;-   2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene;-   9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenol;    and    9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-bromofluorene.    2. Production Method of the Compound of the Present Invention

While the production method of the compounds of the present invention isnot particularly limited, for example, it can be synthesized by thefollowing reactions.

Unless particularly specified, the starting material compounds may be acommercially available product, or can be produced according to a methodknown per se or a method analogous thereto.

While the yield of the compound obtained by each of the followingmethods may vary depending on the reaction conditions employed, theseresultant products can be isolated and purified by a general method(recrystallization, column chromatography and the like), and thenprecipitated by a method of changing the solution temperature, a methodof changing the solution composition and the like.

In each reaction, when the starting material compound has a hydroxygroup, an amino group, a carboxy group or a carbonyl group, a protectinggroup generally used in the peptide chemistry and the like may beintroduced into these groups, and the object compound can be obtained byremoving the protecting group as necessary after the reaction.

Examples of the hydroxyl-protecting group include (C₁-C₆)alkyl group(e.g., methyl, ethyl, propyl, isopropyl, butyl, tert-butyl), phenylgroup, trityl group, (C₇-C₁₀)aralkyl group (e.g., benzyl), formyl group,(C₁-C₆)alkyl-carbonyl group (e.g., acetyl, propionyl), benzoyl group,(C₇-C₁₀)aralkyl-carbonyl group (e.g., benzylcarbonyl),2-tetrahydropyranyl group, 2-tetrahydrofuranyl group, silyl group (e.g.,trimethylsilyl, triethylsilyl, dimethylphenylsilyl,tert-butyldimethylsilyl, tert-butyldiethylsilyl), (C₂-C₆)alkenyl group(e.g., 1-allyl) and the like. These groups are optionally substituted by1 to 3 substituents selected from a halogen atom (e.g., fluorine,chlorine, bromine, iodine), a (C₁-C₆)alkyl group (e.g., methyl, ethyl,propyl), a (C₁-C₆)alkoxy group (e.g., methoxy, ethoxy, propoxy), a nitrogroup and the like.

Examples of the amino-protecting group include formyl group,(C₁-C₆)alkyl-carbonyl group (e.g., acetyl, propionyl),(C₁-C₆)alkoxy-carbonyl group (e.g., methoxycarbonyl, ethoxycarbonyl,tert-butoxycarbonyl), benzoyl group, (C₇-C₁₀)aralkyl-carbonyl group(e.g., benzylcarbonyl), (C₇-C₁₄)aralkyloxy-carbonyl group (e.g.,benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl), trityl group, phthaloylgroup, N,N-dimethylaminomethylene group, silyl group (e.g.,trimethylsilyl, triethylsilyl, dimethylphenylsilyl,tert-butyldimethylsilyl, tert-butyldiethylsilyl), (C₂-C₆)alkenyl group(e.g., 1-allyl) and the like. These groups are optionally substituted by1 to 3 substituents selected from a halogen atom (e.g., fluorine,chlorine, bromine, iodine), a (C₁-C₆)alkoxy group (e.g., methoxy,ethoxy, propoxy), a nitro group and the like.

Examples of the carboxy-protecting group include (C₁-C₆)alkyl group(e.g., methyl, ethyl, propyl, isopropyl, butyl, tert-butyl),(C₇-C₁₀)aralkyl group (e.g., benzyl), phenyl group, trityl group, silylgroup (e.g., trimethylsilyl, triethylsilyl, dimethylphenylsilyl,tert-butyldimethylsilyl, tert-butyldiethylsilyl,tert-butyldiphenylsilyl), (C₂-C₆)alkenyl group (e.g., 1-allyl) and thelike can be mentioned. These groups are optionally substituted by 1 to 3substituents selected from a halogen atom (e.g., fluorine, chlorine,bromine, iodine), a (C₁-C₆)alkoxy group (e.g., methoxy, ethoxy,propoxy), a nitro group and the like.

Examples of the carbonyl-protecting group include cyclic acetal (e.g.,1,3-dioxane), acyclic acetal (e.g., di-(C₁-C₆)alkylacetal) and the like.

These protecting groups can be removed by a method known per se, forexample, the method described in Protective Groups in Organic Synthesis,John Wiley and Sons (1980) and the like. For example, a method usingacid, base, ultraviolet rays, hydrazine, phenylhydrazine, sodiumN-methyldithiocarbamate, tetrabutylammonium fluoride, palladium acetate,trialkylsilylhalide (e.g., trimethylsilyliodide, trimethylsilylbromideand the like) and the like, a reduction method and the like are used.

Production Method

An example wherein at least any one of ring A, ring B and ring C(hereinafter also to be referred simply as a ring) is substituted by anorganic group having an aliphatic hydrocarbon group via —O— is shown inthe following.

In this method, as the “aliphatic hydrocarbon group”, those similar tothe “aliphatic hydrocarbon group” of the above-mentioned “organic grouphaving an aliphatic hydrocarbon group” can be mentioned.

Step 1.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably bromine atom), and ALK¹ and ALK² are the sameor different and each is an aliphatic hydrocarbon group.

Compound (1) is reacted with 0.2 to 5 molar equivalents of compound (2)to give compound (3). Generally, the reaction is performed in thepresence of a base in a solvent that does not adversely influence thereaction.

Examples of the base include an alkali metal salt such as potassiumhydroxide, sodium hydroxide, sodium hydrogen carbonate, potassiumcarbonate and the like; amine such as pyridine, triethylamine,N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-en and the like;metal hydride such as potassium hydride, sodium hydride and the like;alkali metal alkoxide such as sodium methoxide, sodium ethoxide,potassium tert-butoxide and the like, and the like.

The amount of the base to be used is preferably about 1 to 5 molarequivalents relative to compound (1).

As such solvent, halogenated hydrocarbon such as chloroform,dichloromethane and the like; and nonpolar organic solvent such as1,4-dioxane, tetrahydrofuran and the like can be mentioned. Thesesolvents may be used in a mixture of two or more kinds at an appropriateratio. It is preferably tetrahydrofuran. The amount of the solvent to beused is generally 2- to 50-fold volume relative to compound (1).

The reaction temperature is generally 20° C. to 150° C., preferably 50°C. to 100° C. The reaction time is generally 1 to 30 hours.

Step 2.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably bromine atom), and ALK² and ALK³ are the sameor different and each is an aliphatic hydrocarbon group.

Compound (4) is reacted with about 0.3 to 5 molar equivalents ofcompound (2) to give compound (5). Generally, the reaction is performedin the presence of a base in a solvent that does not adversely influencethe reaction.

Examples of the base include an alkali metal salt such as potassiumhydroxide, sodium hydroxide, sodium hydrogen carbonate, potassiumcarbonate and the like; amine such as pyridine, triethylamine,N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-en and the like;metal hydride such as potassium hydride, sodium hydride and the like;alkali metal alkoxide such as sodium methoxide, sodium ethoxide,potassium tert-butoxide and the like, and the like.

The amount of the base to be used is preferably about 1 to 10 molarequivalents relative to compound (4).

As such solvent, halogenated hydrocarbon such as chloroform,dichloromethane and the like; and nonpolar organic solvent such as1,4-dioxane, tetrahydrofuran and the like can be mentioned. Thesesolvents may be used in a mixture of two or more kinds at an appropriateratio. It is preferably tetrahydrofuran. The amount of the solvent to beused is generally 2- to 50-fold volume relative to compound (2).

The reaction temperature is generally 20° C. to 150° C., preferably 50°C. to 100° C. The reaction time is generally 1 to 30 hours.

Step 3-1.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably bromine atom) and ALK⁴ is an aliphatichydrocarbon group.

Compound (6) is reacted with 0.3 to 5 molar equivalents of compound (7)to give compound (8). Generally, the reaction is performed in thepresence of a base in a solvent that does not adversely influence thereaction.

Examples of the base include an alkali metal salt such as potassiumhydroxide, sodium hydroxide, sodium hydrogen carbonate, potassiumcarbonate and the like; amine such as pyridine, triethylamine,N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-en and the like;metal hydride such as potassium hydride, sodium hydride and the like;alkali metal alkoxide such as sodium methoxide, sodium ethoxide,potassium tert-butoxide and the like, and the like.

The amount of the base to be used is preferably about 1 to 5 molarequivalents relative to compound (6).

Examples of the solvent include aromatic hydrocarbon such as benzene,toluene, xylene and the like; nitrile such as acetonitrile,propionitrile and the like; ether such as tetrahydrofuran, 1,4-dioxane,diethyl ether and the like; ketone such as acetone, 2-butanone and thelike; halogenated hydrocarbon such as chloroform, dichloromethane andthe like; amide such as N,N-dimethylformamide and the like; sulfoxidesuch as dimethyl sulfoxide and the like, and the like. These solventsmay be used in a mixture of two or more kinds at an appropriate ratio.It is preferably dimethylformamide. The amount of the solvent to be usedis generally 2- to 50-fold volume relative to compound (6).

The reaction temperature is generally 30° C. to 150° C., preferably 50°C. to 100° C. The reaction time is generally 1 to 30 hours.

Step 3-2.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably bromine atom) and ALK⁴ and ALK^(4′) are thesame or different and each is an aliphatic hydrocarbon group.

Compound (6′) is reacted with halide to give compound (8′). Whencompound (7) and compound (7′) are the same, the above-mentionedreaction can be performed in one step. When compound (7) and compound(7′) are different compounds, compound (6′) is reacted with 0.3 to 5molar equivalents of compound (7) and then reacted with 0.3 to 5 molarequivalents of compound (7′). During reaction with compound (7), themoiety to be reacted with compound (7′) is preferably protected with aprotecting group in advance. As the protecting group,hydroxyl-protecting group can be mentioned and, for example,(C₁-C₆)alkyl group (e.g., methyl, ethyl, propyl, isopropyl, butyl,tert-butyl), phenyl group, trityl group, (C₇-C₁₀)aralkyl group (e.g.,benzyl), formyl group, (C₁-C₆)alkyl-carbonyl group (e.g., acetyl,propionyl), benzoyl group, (C₇-C₁₀)aralkyl-carbonyl group (e.g.,benzylcarbonyl), 2-tetrahydropyranyl group, 2-tetrahydrofuranyl group,silyl group (e.g., trimethylsilyl, triethylsilyl, dimethylphenylsilyl,tert-butyldimethylsilyl, tert-butyldiethylsilyl), (C₂-C₆)alkenyl group(e.g., 1-allyl) and the like can be mentioned.

These reactions are generally performed in the presence of a base in asolvent that does not adversely influence the reaction.

Examples of the base include an alkali metal salt such as potassiumhydroxide, sodium hydroxide, sodium hydrogen carbonate, potassiumcarbonate and the like; amine such as pyridine, triethylamine,N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-en and the like;metal hydride such as potassium hydride, sodium hydride and the like;alkali metal alkoxide such as sodium methoxide, sodium ethoxide,potassium tert-butoxide and the like, and the like.

The amount of the base to be used is preferably about 1 to 10 molarequivalents relative to compound (6′).

Examples of the solvent include aromatic hydrocarbon such as benzene,toluene, xylene and the like; nitrile such as acetonitrile,propionitrile and the like; ether such as tetrahydrofuran, 1,4-dioxane,diethyl ether and the like; ketone such as acetone, 2-butanone and thelike; halogenated hydrocarbon such as chloroform, dichloromethane andthe like; amide such as N,N-dimethylformamide and the like; sulfoxidesuch as dimethyl sulfoxide and the like, and the like. These solventsmay be used in a mixture of two or more kinds at an appropriate ratio.It is preferably dimethylformamide. The amount of the solvent to be usedis generally 2- to 50-fold volume relative to compound (6′).

The reaction temperature is generally 30° C. to 150° C., preferably 50°C. to 100° C. The reaction time is generally 1 to 70 hours.

Step 3-3.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably bromine atom) and ALK² and ALK³ are the sameor different and each is an aliphatic hydrocarbon group.

Compound (6) reacted with about 0.5 to 5 molar equivalents of compound(5) to give compound (9). Generally, the reaction is performed in thepresence of a base in a solvent that does not adversely influence thereaction.

Examples of the base include an alkali metal salt such as potassiumhydroxide, sodium hydroxide, sodium hydrogen carbonate, potassiumcarbonate and the like; amine such as pyridine, triethylamine,N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-en and the like;metal hydride such as potassium hydride, sodium hydride and the like;alkali metal alkoxide such as sodium methoxide, sodium ethoxide,potassium tert-butoxide and the like, and the like.

The amount of the base to be used is preferably about 1 to 10 molarequivalents relative to compound (6).

Examples of the solvent include aromatic hydrocarbon such as benzene,toluene, xylene and the like; nitrile such as acetonitrile,propionitrile and the like; ether such as tetrahydrofuran, 1,4-dioxane,diethyl ether and the like; ketone such as acetone, 2-butanone and thelike; halogenated hydrocarbon such as chloroform, dichloromethane andthe like; amide such as N,N-dimethylformamide and the like; sulfoxidesuch as dimethyl sulfoxide and the like, and the like. These solventsmay be used in a mixture of two or more kinds at an appropriate ratio.It is preferably dimethylformamide. The amount of the solvent to be usedis generally 2- to 50-fold volume relative to compound (6).

The reaction temperature is generally 30° C. to 150° C., preferably 50°C. to 100° C. The reaction time is generally 1 to 70 hours.

Step 4.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably chlorine atom), and ALK⁴ is an aliphatichydrocarbon group.

Compound (8) is reacted with a Grignard reagent generally in a solventthat does not adversely influence the reaction to give compound (I-1).

As the Grignard reagent used in this step, 4-chlorophenylmagnesiumbromide, 3-chlorophenylmagnesium bromide,3-trifluoromethylphenylmagnesium bromide, 3-fluorophenylmagnesiumbromide, 3,5-difluorophenylmagnesium bromide and the like can bementioned.

The amount of the Grignard reagent to be used is preferably about 1 to10 molar equivalents relative to compound (8).

As such solvent, halogenated hydrocarbon such as chloroform,dichloromethane and the like; and nonpolar organic solvent such as1,4-dioxane, tetrahydrofuran and the like can be mentioned. Thesesolvents may be used in a mixture of two or more kinds at an appropriateratio. It is preferably tetrahydrofuran. The amount of the solvent to beused is generally 2- to 50-fold volume relative to compound (8).

The reaction temperature is generally 10° C. to 100° C., preferably 30°C. to 70° C. The reaction time is generally 1 to 30 hours.

Step 5.

wherein Hal₁ is a bromine atom or a chlorine atom, Hal is a halogen atom(chlorine atom, bromine atom, iodine atom, fluorine atom; preferablychlorine atom) and ALK⁴ is an aliphatic hydrocarbon group.

Compound (I-1) is reacted with a halogenating agent generally in asolvent that does not adversely influence the reaction to give compound(I-2).

As the halogenating agent to be used in this step, acetyl bromide,acetyl chloride, thionyl chloride, thionyl bromide, PBr₃, HBr, NBS andthe like can be mentioned.

The amount of halogenated acyl to be used is preferably about 1 to 30molar equivalents relative to compound (I-1).

As such solvent, halogenated hydrocarbon such as chloroform,dichloromethane and the like; and nonpolar organic solvent such as1,4-dioxane, tetrahydrofuran and the like can be mentioned. Thesesolvents may be used in a mixture of two or more kinds at an appropriateratio. It is preferably chloroform. The amount of the solvent to be usedis generally 2- to 50-fold volume relative to compound (I-1).

The reaction temperature is generally 10° C. to 100° C., preferably 50°C. to 80° C. The reaction time is generally 1 to 70 hours.

Compound (I-3) can be obtained by successively performing the reactionsin step 4 and step 5 using compound (3) obtained in step 1 instead ofcompound (7) in step 3-1.

wherein Hal₁ is a bromine atom or a chlorine atom, Hal is a halogen atom(chlorine atom, bromine atom, iodine atom, fluorine atom; preferablyfluorine atom), and ALK¹ and ALK² are the same or different and each isan aliphatic hydrocarbon group.Step 6.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably fluorine atom), ALK⁴ is an aliphatichydrocarbon group, X₂″ in the number of m₂ are each independentlyabsent, or —O—, —S—, —NHCO— or —CONH—, n₁, n₂, n₃ and n₄ are eachindependently an integer of 0 to 2, and m₂ is an integer of 1 or 2, whena plurality of n₁, n₂, n₃ and n₄ are each present, each respective n₁,n₂, n₃ and n₄ may be the same or different, and when a plurality of ALK⁴is present, to respective ALK⁴ may be the same or different.

Compound (7) is reacted with 0.2 to 5 molar equivalents of compound (10)to give compound (11). Generally, the reaction is performed in thepresence of a base in a solvent that does not adversely influence thereaction.

Examples of the base include an alkali metal salt such as potassiumhydroxide, sodium hydroxide, sodium hydrogen carbonate, potassiumcarbonate and the like; amine such as pyridine, triethylamine,N,N-dimethylaniline, 1,8-diazabicyclo[5.4.0]undec-7-en and the like;metal hydride such as potassium hydride, sodium hydride and the like;alkali metal alkoxide such as sodium methoxide, sodium ethoxide,potassium tert-butoxide and the like, and the like.

The amount of the base to be used is preferably about 1 to 5 molarequivalents relative to compound (7).

Examples of the solvent include aromatic hydrocarbon such as benzene,toluene, xylene and the like; nitrile such as acetonitrile,propionitrile and the like; ether such as tetrahydrofuran, 1,4-dioxane,diethyl ether and the like; ketone such as acetone, 2-butanone and thelike; halogenated hydrocarbon such as chloroform, dichloromethane andthe like; amide such as N,N-dimethylformamide and the like; sulfoxidesuch as dimethyl sulfoxide and the like, and the like. These solventsmay be used in a mixture of two or more kinds at an appropriate ratio.It is preferably dimethylformamide. The amount of the solvent to be usedis generally 2- to 50-fold volume relative to compound (7).

The reaction temperature is generally 20° C. to 150° C., preferably 50°C. to 100° C. The reaction time is generally 1 to 30 hours.

Step 7.

wherein Hal is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom; preferably iodine atom), ALK⁴ is an aliphatic hydrocarbongroup, X₂″ in the number of m₂ are each independently absent, or —O—,—S—, —NHCO— or —CONH—, n₁, n₂, n₃ and n₄ are each independently aninteger of 0 to 2, and m₂ is an integer of 1 or 2, when a plurality ofn₁, n₂, n₃ and n₄ are each present, each respective n₁, n₂, n₃ and n₄may be the same or different, and when a plurality of ALK⁴ is present,respective ALK⁴ may be the same or different.

A hydroxyl group of compound (11) is substituted by halogen to givecompound (11′).

Triphenylphosphine and a halogen source are reacted in a solvent thatdoes not adversely influence the reaction. Generally, the reaction ispreferably performed in the presence of imidazole. As the halogensource, carbon tetrachloride, hexachloroacetone and triphosgene(chlorine source), carbon tetrabromide (bromine source), iodomethane andiodine (iodine source) and the like can be mentioned. The amount oftriphenylphosphine to be used is preferably about 0.1-5 molarequivalents relative to compound (11), and the amount of the halogensource to be used is about 1 to 5 molar equivalents relative to compound(11). When imidazole is used, the amount thereof to be used is about 0.1to 5 molar equivalents relative to compound (11).

Examples of the solvent include aromatic hydrocarbon such as benzene,toluene, xylene and the like; nitrile such as acetonitrile,propionitrile and the like; ether such as tetrahydrofuran, 1,4-dioxane,diethyl ether and the like; ketone such as acetone, 2-butanone and thelike; halogenated hydrocarbon such as chloroform, dichloromethane andthe like; amide such as N,N-dimethylformamide and the like; sulfoxidesuch as dimethyl sulfoxide and the like, and the like. These solventsmay be used in a mixture of two or more kinds at an appropriate ratio.It is preferably toluene. The amount of the solvent to be used isgenerally 3-50-fold volume relative to compound (11).

The reaction temperature is generally 30° C. to 150° C., preferably 40°C. to 120° C. The reaction time is generally 0.5 to 24 hours.

Compound (11) and compound (11′) produced via step 6 and step 7 can be auseful intermediate to produce the compound of the present invention.

Using compounds (11) and (11′) obtained in step 6 and step 7, anintermediate useful for producing the compound of the present inventioncan be obtained by performing the reactions of the above-mentioned step3-1, step 3-2 and/or step 3-3. One example is shown in the following. Inthe scheme, the carbon number of aliphatic hydrocarbon group, the kindof halogen atom, reaction reagent and the like are shown forconvenience, and can be appropriately changed within the range of theabove-mentioned definitions.

-   Ts: tosyl group,-   Ph: phenyl group,-   Py: pyridine,-   Et: ethyl group

wherein X is a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom).

In addition, even when “an organic group having an aliphatic hydrocarbongroup” is the following formula (d):

wherein each symbol is as defined above, an intermediate useful forproducing the compound of the present invention can be obtained byperforming the reactions of the above-mentioned step 3-1, step 3-2and/or step 3-3. One example is shown in the following. In the scheme,the carbon number of aliphatic hydrocarbon group, the kind of halogenatom, reaction reagent and the like are shown for convenience, and canbe appropriately changed within the range of the above-mentioneddefinitions.

In addition, even when “an organic group having an aliphatic hydrocarbongroup” is the following formula (e):

wherein each symbol is as defined above, an intermediate useful forproducing the compound of the present invention can be obtained byperforming the reactions of the above-mentioned step 3-1, step 3-2and/or step 3-3. One example is shown in the following. In the scheme,the carbon number of aliphatic hydrocarbon group, the kind of halogenatom, reaction reagent and the like are shown for convenience, and canbe appropriately changed within the range of the above-mentioneddefinitions.

When a compound wherein an organic group having an aliphatic hydrocarbongroup substitutes a ring via a group other than —O— is to be produced,the compound of the present invention can be produced according to theabove-mentioned method, or by appropriately changing the above-mentionedmethod. Such change can be made by performing a reaction generallyperformed in the art. For example, an organic group having an aliphatichydrocarbon group substitutes a ring via —NH— can be obtained bydehydrative condensation of a compound having a carboxyl group on endand a compound having an amino group on end.

3. Organic Synthesis Reaction Method

The compound of the present invention can be used as a protectingreagent for various organic synthesis reactions. For example, thefollowing steps are performed.

(i) a step of binding the compound of the present invention to aminoacid or peptide (binding step), and

(ii) a step of precipitating the bonded product of the compound and theamino acid or peptide obtained in the above-mentioned step(precipitation step).

First, the compound of the present invention is dissolved in a solublesolvent, then a reaction substrate to be reacted (amino acid or peptideto be a material for peptide synthesis here) is added to a reagentdissolved in a soluble solvent to bind the both (binding step). As asolvent to be used for the reaction system, a general organic solventcan be used. Since superior reactivity can be expected when thesolubility in the solvent is higher, a solvent in which the compound ofthe present invention shows high solubility is preferably selected.Specifically, halogenated hydrocarbon such as chloroform,dichloromethane and the like; and a nonpolar organic solvent such as1,4-dioxane, tetrahydrofuran and the like can be mentioned. Thesesolvents may be used in a mixture of two or more kinds at an appropriateratio. In addition, the above-mentioned halogenated hydrocarbons andnonpolar organic solvent may be mixed with aromatic hydrocarbon such asbenzene, toluene, xylene and the like; nitrile such as acetonitrile,propionitrile and the like; ketone such as acetone, 2-butanone and thelike; amide such as N,N-dimethylformamide and the like; sulfoxide suchas dimethyl sulfoxide and the like at an appropriate ratio and used aslong as the compound of the present invention is dissolved.

For confirmation of the progress of the reaction, a method similar togeneral liquid phase organic synthesis reaction can be applied. That is,thin layer silica gel chromatography, high performance liquidchromatography and the like can be used to track the reaction.

Step i (Binding Step)

In this step, the compound of the present invention dissolved in asoluble solvent is bonded to a reactive substrate.

wherein Hal₁ is a bromine atom or a chlorine atom, Hal is a halogen atom(chlorine atom, bromine atom, iodine atom, fluorine atom; preferablyfluorine atom), P is an amino-protecting group, AA is a group derivedfrom amino acid, and ALK⁴ is an aliphatic hydrocarbon group.

The compound of the present invention (I-2) is reacted with a protectedamino acid (amino acid bound with a protecting group: P-AA-OH) generallyin a solvent that does not adversely influence the reaction to give acompound (bonded product) wherein compound (I-2) is bound with protectedamino acid. The amino acid of the “group derived from amino acid” is notparticularly limited as long as it is intended to be bonded to compound(I-2), and may be natural amino acid or non-natural amino acid.

The compound of the present invention such as compound (I-2) and thelike is bonded to amino acid or peptide to be a starting material andfunctions as an anchor in a peptide synthesis reaction.

While the protecting group of protected amino acid (corresponding to Pin the above-mentioned scheme) varies, for example, by protection targetamino acid, those generally used, specifically those similar to theabove-mentioned groups can be recited as examples. When amino acid isalanine, P is preferably a benzyloxycarbonyl (Z) group or a9-fluorenylmethoxycarbonyl (Fmoc) group.

The protected amino acid used in this step is of the same kind as theamino acid to be bonded to compound (I-2). For example, when alanine isto be bonded to (I-2), the protected amino acid is P-alanine.

The amount of protected amino acid to be used in this step is preferablyabout 1 to 10 molar equivalents relative to compound (I-2).

As such solvent, halogenated hydrocarbon such as chloroform,dichloromethane and the like; and nonpolar organic solvent such as1,4-dioxane, tetrahydrofuran and the like can be mentioned. Thesesolvents may be used in a mixture of two or more kinds at an appropriateratio. It is preferably chloroform. The amount of the solvent to be usedis generally 2- to 50-fold volume relative to compound (I-2).

The reaction temperature is generally 10° C. to 100° C., preferably 20°C. to 70° C. The reaction time is generally 1 to 70 hours.

When a compound of the present invention wherein Y is a hydroxyl groupis used, the reaction substrate is a compound having an amino group, andwhen a compound of the present invention wherein Y is an amino group isused, the reaction substrate is a compound having a carboxyl group.

Step ii (Precipitation Step)

In this step, the solvent used to dissolve the bonded product obtainedin the above-mentioned step is changed to cause precipitation to isolatethe bonded product.

After the reaction, the solvent used to dissolve the compound is changed(e.g., change of solvent composition, change of solvent kind) toconveniently isolate the obtained bonded product by precipitation. Thatis, the bonded product is precipitated and impurity is eliminated byperforming a reaction under conditions where the compound is dissolved,and changing the solvent after the reaction. Examples of the change ofsolvent include use of a halogen solvent, THF and the like fordissolution, and a polar organic solvent such as methanol, acetonitrileand the like for precipitation.

The bonded product isolated by precipitation is subjected to a desiredreaction, and protecting reagent derived from the compound of thepresent invention is finally removed (deprotection step).

For removal of the protecting reagent, various methods generally used inthe art, particularly in the peptide synthesis, are employed. Generally,a method including addition of an acid and the like is employed. As theacid, trifluoroacetic acid (TFA), hydrochloric acid, sulfuric acid,mesylic acid, tosylic acid, trifluoroethanol, hexafluoroisopropanol andthe like are used. Among these, TFA is preferable.

The amount of the acid to be used is appropriately determined accordingto the kind of the acid, and an amount suitable for removing theprotecting reagent is used. The reaction temperature is generally 0° C.to 80° C., preferably 10° C. to 50° C. The reaction time is generally0.5 to 24 hours.

Peptide can be produced by liquid phase synthesis utilizing theabove-mentioned steps. Specifically, the synthesis includes thefollowing steps.

(1) a step of condensing the fluorene compound of the present inventionwith the C-terminal of N-protected amino acid or N-protected peptide togive C-fluorene compound-protected amino acid or C-fluorenecompound-protected peptide (C-terminal fluorene compound protectionstep),

(2) a step of removing the protecting group from the N-terminal of theamino acid or peptide obtained in the above-mentioned step (N-terminaldeprotection step),

(3) a step of condensing the N-terminal of the amino acid or peptideobtained in the above-mentioned step with N-protected amino acid orN-protected peptide (peptide chain elongation step), and

(4) a step of precipitating the peptide obtained in the above-mentionedstep (precipitation step).

Step 1 (C-Terminal Fluorene Compound Protection Step)

In this step, the fluorene compound of the present invention iscondensed with the C-terminal of N-protected amino acid or N-protectedpeptide to give C-fluorene compound-protected amino acid or C-fluorenecompound-protected peptide. For example, the step can be performedaccording to the above-mentioned binding step.

In the present invention, the “N-protected amino acid” and “N-protectedpeptide” mean amino acid and peptide wherein an amino group is protectedand a carboxyl group is not protected, which can be indicated as“P-AA-OH” (P is an amino-protecting group).

The condensation reaction of the fluorene compound of the presentinvention with N-protected amino acid or N-protected peptide at theC-terminal is generally performed in a solvent that does not influencethe reaction. For example, when Y is a halogen atom (e.g., a bromogroup, a chloro group, an iodo group, preferably a bromo group and achloro group), the reaction can be performed in the presence of a base.As the base, etheramine, ethylamine, trimethylamine, triethylamine,triethanolamine, diisopropylethylamine and the like can be mentioned.When Y is a hydroxyl group, the reaction can be performed in thepresence of a condensing agent. Examples of the condensing agent includedicyclohexylcarbodiimide, diisopropylcarbodiimide,N-ethyl-N′-3-dimethylaminopropylcarbodiimide and hydrochloride thereof(EDC-HCl) and the like. Where necessary, a condensing agent may be usedtogether with a promoter such as N-hydroxysuccinimide,1-hydroxybenzotriazole (HOBt) and the like.

As the solvent to be used for this step, halogenated hydrocarbon such aschloroform, dichloromethane and the like; and nonpolar organic solventsuch as 1,4-dioxane, tetrahydrofuran and the like can be mentioned.These solvents may be used in a mixture of two or more kinds at anappropriate ratio. It is preferably chloroform. The amount of thesolvent to be used is generally 2- to 50-fold volume relative to thefluorene compound of the present invention.

The reaction temperature is generally 10° C. to 100° C., preferably 20°C. to 70° C. The reaction time is generally 1 to 70 hours.

Step 2 (N-Terminal Deprotection Step)

In this step, the protecting group at the N-terminal of the amino acidor peptide obtained in the above-mentioned step is removed.

Deprotection is appropriately selected according to the kind of theN-protecting group. A group that can be removed under conditionsdifferent from those for the removal of the protecting reagent derivedfrom the compound of the present invention is preferable. For example,for an Fmoc group, it is removed by treating with a base. The reactionis generally performed in a solvent that does not influence thereaction.

As the base, dimethylamine, diethylamine and the like can be mentioned.Examples of the solvent include halogenated hydrocarbon such aschloroform, dichloromethane and the like; aromatic hydrocarbon such astoluene, xylene and the like; ether such as diethyl ether,tetrahydrofuran, dioxane and the like; nitrile such as acetonitrile andthe like, and a mixture thereof can be mentioned.

Step 3 (Peptide Chain Elongation Step)

In this step, the N-terminal of amino acid or peptide deprotected instep 2 is condensed with N-protected amino acid or N-protected peptide.

This step is performed according to the method of the above-mentionedstep 1 wherein Y is a hydroxyl group.

Step 4 (Precipitation Step)

This step is performed in the same manner as in the precipitation stepin the above-mentioned step ii.

In the method of producing a peptide of the present invention,N-protected amino acid or protected peptide obtained in step 4 can besubjected to a desired number of repeats of steps (5)-(7).

(5) a step of deprotecting the N-terminal of the peptide obtained in theprecipitation step,

(6) a step of condensing the N-terminal of peptide obtained in theabove-mentioned step with N-protected amino acid or N-protected peptide,and

(7) a step of precipitating the peptide obtained in the above-mentionedstep.

Step 5

This step is performed in the same manner as in the N-terminaldeprotection step of step 2.

Step 6

This step is performed in the same manner as in the peptide chainelongation step of step 3.

Step 7

This step is performed in the same manner as in the precipitation stepof step ii.

The method of producing a peptide of the present invention can furthercomprise, after the precipitation step of step 4 or step 7, a step ofdeprotecting the C-terminal of peptide which is protected with afluorene compound. For example, the step is performed according to thestep of removing the protecting reagent of the present inventionmentioned above.

4. Kit for Liquid Phase Synthesis of Peptide

The present invention also provides a kit for liquid phase synthesis ofpeptide, which contains the above-mentioned compound of the presentinvention as an essential constituent component. The kit may contain,besides the compound of the present invention, other componentsnecessary for liquid phase synthesis reaction of peptide, for example,various solvents used for the reaction, amino acid (or peptide) to bethe starting material and the like. When desired, a manual of liquidphase synthesis of peptide using the compound of the present inventioncan also be attached.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES

The reagents, apparatuses and materials used in the present inventionare commercially available unless otherwise specified. In the presentspecification, when amino acid and the like are indicated byabbreviation, each indication is based on the abbreviation of theIUPAC-IUB Commission on Biochemical Nomenclature or conventionalabbreviations in the art.

Example 1 1-1: Synthesis of 2-docosyloxy-9-fluorenone

2-Hydroxy-9-fluorenone (196 mg, 2.04 mmol) was dissolved in DMF (8 ml),potassium carbonate (423 mg, 3.06 mmol), and docosyl bromide (96%, 785mg, 1.93 mmol) were added. The mixture was heated to 80° C. and stirredovernight. After completion of the reaction, the reaction mixture wascooled to room temperature, and 1N hydrochloric acid (4 ml) was addeddropwise thereto in a water bath to quench the reaction. The mixture wasextracted with chloroform (23 ml), and washed once with 1N hydrochloricacid (7.5 ml) and 4 times with pure water (7.5 ml). The solvent of theorganic layer was evaporated, and the residue was precipitated withmethanol (10 ml) to give 2-docosyloxy-9-fluorenone (934 mg, 1.85 mmol,96%).

¹H-NMR(400 MHz)

0.88 (3H, t, J=7.0, C₂₁H₄₂-Me) 1.20-1.40 (36H, br, alkyl-H) 1.45 (2H,br, —O—C₂H₄—CH₂ —C₁₉H₃₉) 1.79 (2H, m, —O—CH₂—CH₂ —) 4.00 (2H, t, J=6.6,—O—CH₂ —) 6.97 (1H, fluorenone C3-H) 7.19 (2H, fluorenone C1,7-H)7.38-7.43 (3H, fluorenone C4,5,6-H) 7.59 (1H, fluorenone C8-H)

MS

505 [M+H]

1-2: Synthesis of 2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol

Under a nitrogen atmosphere, 2-docosyloxy-9-fluorenone (250 mg, 0.50mmol) prepared in the above-mentioned 1-1 was suspended in THF (2.5 ml),and dissolved at 50° C. 4-Chlorophenylmagnesium bromide solution wasadded dropwise, and the mixture was stirred. After completion of thereaction, the reaction mixture was cooled to room temperature, 1Nhydrochloric acid was added dropwise thereto in a water bath untilfoaming stopped to quench the reaction. The mixture was extracted withchloroform (15 ml), and washed 3 times with 1N hydrochloric acid (5 ml),3 times with 5% NaHCO₃ solution (5 ml), and once with 20% NaCl solution(5 ml). The organic layer was dried over Na₂SO₄, and the solvent of thefiltrate was evaporated. The obtained residue was isolated and purifiedby silica gel column chromatography, and precipitated with methanol (3ml) to give 2-docosyloxy-9-(4-chlorophenyl)-9-fluorenol (270 mg, 0.44mmol, 88%).

¹H-NMR(400 MHz)

0.88 (3H, t, J=7.0, C₂₁H₄₂-Me) 1.20-1.40 (36H, br, alkyl-H) 1.45 (2H,br, —O—C₂H₄—CH₂ —C₁₉H₃₉) 1.75 (2H, m, —O—CH₂—CH₂ —) 2.39 (1H, s, OH)3.91 (2H, —O—CH₂ —) 6.82 (1H, fluorene C3-H) 6.89 (1H, fluorene C1-H)7.18 (1H, fluorene C7-H) 7.21-7.35 (6H, Ph-H, fluorenone-H) 7.56 (2H,fluorenone-H)

MS

599 [M-OH]

1-3: Synthesis of 2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene

2-Docosyloxy-9-(4-chlorophenyl)-9-fluorenol (200 mg, 0.33 mmol) preparedin the above-mentioned 1-2 was dissolved in chloroform (2 ml), acetylbromide (48 μl, 0.65 mmol) was added dropwise and the mixture wasstirred for 1 hour. After completion of the reaction, the solvent wasevaporated, and the residue was precipitated with acetonitrile (2 ml) togive 2-docosyloxy-9-(4-chlorophenyl)-9-bromofluorene (209 mg, 0.31 mmol,95%).

¹H-NMR(400 MHz)

0.88 (3H, t, J=7.0, C₂₁H₄₂-Me) 1.20-1.40 (36H, br, alkyl-H) 1.44 (2H,br, —O—C₂H₄—CH₂ —C₁₉H₃₉) 1.77 (2H, m, —O—CH₂—CH₂ —) 3.95 (2H, —O—CH₂ —)6.92 (1H, fluorene-H) 6.98 (1H, fluorene-H) 7.21-7.26 (3H) 7.34 (1H)7.43 (1H) 7.46-7.49 (2H) 7.55-7.59 (2H)

Example 2 2-1: Synthesis of 2,7-didocosyloxy-9-fluorenone

2,7-Dihydroxy-9-fluorenone (1 g, 4.71 mmol) was dissolved in DMF (40ml), and potassium carbonate (1.95 g, 14.1 mmol) and docosyl bromide(96%, 4.02 g, 9.91 mmol) were added. The mixture was stirred overnightat 80° C., docosyl bromide (0.40 g, 0.99 mmol) was further added and themixture was stirred overnight. After completion of the reaction, thereaction mixture was cooled to room temperature, 1N hydrochloric acid(120 ml) was added thereto in a water bath to allow precipitation. Thecrystals were collected by filtration and slurry washed once with 1Nhydrochloric acid (40 ml), once with pure water (40 ml) and once withmethanol (40 ml) to give 2,7-didocosyloxy-9-fluorenone (4.20 g, docosylbromide mixture).

¹H-NMR(300 MHz)

0.88 (6H, t, J=6.6, C₂₁H₄₂-Me) 1.15-1.60 (80H, br, alkyl-H) 1.78 (4H, m,—O—CH₂—CH₂ —) 3.98 (4H, t, J=6.6, —O—CH₂ —) 6.92 (2H, fluorenone C3,6-H)7.14 (2H, d, J=2.1, fluorenone C1,8-H) 7.26 (2H, d, J=7.8, fluorenoneC4,5-H) 7.59 (1H, fluorenone C8-H)

MS

829 [M+H]

2-2: Synthesis of 2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromofluorene

In the same manner as in Example 1, 1-2, then 1-3 except that2,7-didocosyloxy-9-fluorenone prepared in the above-mentioned 2-1 wasused instead of 2-docosyloxy-9-fluorenone,2,7-didocosyloxy-9-(4-chlorophenyl)-9-bromofluorene was obtained.

δ=0.88 (6H, t, OC₂₂H₄₅ C22-H) 1.1-1.6 (76H, br, OC₂₂H₄₅ C3-21-H) 1.75(4H, m, OC₂₂H₄₅ C2-H) 3.92 (4H, m, OC₂₂H₄₅ C1-H) 6.87 (2H,m, fl C3,6-H)6.94 (2H, d, fl C1,8-H) 7.20-7.40 (2H, m, Ph C2,6-H) 7.42-7.49 (4H, m,Ph C3,5-H, fl C4,5-H)

Example 3 3-1: Synthesis of 12-docosyloxy-dodecyl bromide

C₂₂H₄₅OH+Br—C₁₂H₂₄—Br→C₂₂H₄₅—O—C₁₂H₂₄—Br

Sodium hydride (60%, 0.98 g, 24.5 mmol) washed with hexane was suspendedin toluene (16 ml), 1-docosanol (4.00 g, 12.2 mmol) was added,1,12-dibromododecane (8.04 g, 24.5 mmol) was added, and the mixture wasstirred at 85° C. for 2 days. The reaction mixture was cooled to roomtemperature, 1N hydrochloric acid (30 ml) was added dropwise thereto ina water bath to quench the reaction. The mixture was extracted withchloroform (100 ml), and washed 3 times with 1N hydrochloric acid (30ml), 3 times with 5% aqueous sodium hydrogen carbonate solution (30 ml),and once with 20% aqueous sodium chloride solution (30 ml). The organiclayer was dried over sodium sulfate, and the solvent of the filtrate wasevaporated. The residue was isolated and purified by silica gel columnchromatography and precipitated with methanol (70 ml) to give12-docosyloxy-dodecyl bromide (yield 78%).

¹H-NMR(300 MHz)

0.88 (3H, t, J=6.9, C₂₁H₄₂-Me) 1.10-1.65 (76H, br, alkyl-H) 1.85 (2H, m,—CH₂ —CH₂—Br) 3.39 (6H, m, C₂₁H₄₃—CH₂ —O—CH₂—C₁₀H₂₀—CH₂ —Br)

MS

573[M+H], 575[M+H+2]

3-2: Synthesis of 2-(12-docosyloxy-dodecanoxy)-9-fluorenone

In the same manner as in Example 1, 1-1 except that12-docosyloxy-dodecyl bromide prepared in the above-mentioned 3-1 wasused instead of docosyl bromide,2-(12-docosyloxy-dodecanoxy)-9-fluorenone was obtained.

δ=0.88 (3H, t, OC₂₂H₄₅ C22-H) 1.1-1.6 (58H, br, Alkyl-H) 1.77 (2H, m,fl-OCH₂CH₂ —) 3.39 (4H, t, —CH₂ OCH₂ —) 4.00 (2H, t, fl-OCH₂ —) 6.97(1H, m, fl C3-H) 7.18 (2H, m, fl C1,7-H) 7.37-7.45 (3H, m, fl C4,5,6-H)7.60 (1H, d, fl C8-H)

3-3: Synthesis of2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene

In the same manner as in Example 1, 1-2, then 1-3 except that2-(12-docosyloxy-dodecanoxy)-9-fluorenone prepared in theabove-mentioned 3-2 was used instead of 2-docosyloxy-9-fluorenone and3-fluorophenylmagnesium bromide instead of 4-chlorophenylmagnesiumbromide, 2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorenewas obtained.

δ=0.88 (3H, t, OC₂₂H₄₅ C22-H) 1.2-1.6 (58H, br, Alkyl-H) 1.77 (2H, m,fl-OCH₂CH₂ —) 3.38 (4H, t, —CH₂ OCH₂ —) 3.95 (2H, m, fl-OCH₂ —) 6.91(1H, m, fl C3-H) 6.94 (1H, m, fl C1-H) 7.00 (1H, d, Ph C6-H) 7.20-7.38(5H, m, Ph C2,4,5-H, fl C6,7-H) 7.44 (1H, d, fl C8-H) 7.58 (2H, d, flC4,5-H)

Example 4 4-1: Synthesis of 1,12-bis-(12-bromododecyloxy)-dodecane

Br—C₁₂H₂₄—Br+HO—C₁₂H₂₄—OH→Br—C₁₂H₂₄—O—C₁₂H₂₄—O—C₁₂H₂₄—Br

Sodium hydride (60%, 652 mg, 16.3 mmol) washed with hexane was suspendedin toluene (3 ml), 1,12-dodecanediol (1.50 g, 7.41 mmol) was added,1,12-dibromododecane (6.08 g, 18.5 mmol) was added, and the mixture wasstirred at 80° C. for 4 days. The reaction mixture was cooled to roomtemperature, and pure water (30 ml) was added dropwise thereto in awater bath to quench the reaction. The mixture was extracted withchloroform (100 ml), and washed 3 times with 1N hydrochloric acid (30ml), 3 times with 5% aqueous sodium hydrogen carbonate solution (30 ml),and once with 20% aqueous sodium chloride solution (30 ml). The organiclayer was dried over sodium sulfate and the solvent of the filtrate wasevaporated. The residue was isolated and purified by silica gel columnchromatography, and precipitated with methanol (50 ml) to give1,12-bis-(12-bromododecyloxy)-dodecane (yield 56%).

¹H-NMR(300 MHz)

1.20-1.65 (56H, br, alkyl-H) 1.85 (4H, dt, —CH₂ —CH₂—Br) 3.39 (12H, m,—O—CH₂ , —CH₂ —Br)

4-2: Synthesis of 1,12-bis-[12-(2′-O-9-fluorenone)-dodecyloxy]-dodecane

2-Hydroxy-9-fluorenone (577 mg, 2.94 mmol) was dissolved in DMF (10 ml),and potassium carbonate (595 mg, 4.37 mmol) and1,12-bis-(12-bromododecyloxy)-dodecane (1 g, 1.44 mmol) prepared in theabove-mentioned 4-1 were added. The mixture was stirred at 80° C.overnight, 2-hydroxy-9-fluorenone (56 mg, 0.29 mmol) was added, and themixture was stirred overnight. After completion of the reaction, thereaction mixture was cooled to room temperature, and 1N hydrochloricacid (15 ml) was added dropwise thereto in a water bath to quench thereaction. The mixture was extracted with chloroform (50 ml), and washed3 times with 1N hydrochloric acid (15 ml), 3 times with 5% aqueoussodium hydrogen carbonate solution (30 ml) and once with 20% aqueoussodium chloride solution (30 ml). The organic layer was dried oversodium sulfate, the solvent of the filtrate was evaporated, and theresidue was precipitated with methanol (20 ml) to give1,12-bis-[12-(2′-O-9-fluorenone)-dodecyloxy]-dodecane (1.28 g, 96%).

¹H-NMR(300 MHz)

1.20-1.65 (56H, br, alkyl-H) 1.79 (4H, m, fluorenone-O—CH₂—CH₂ —) 3.38(8H, t, J=6.6, —C₁₁H₂₂—CH₂ —O—CH₂ —C₁₁H₂₂—) 4.00 (4H, t, J=6.6,fluorenone-O—CH₂ —) 6.97 (2H, dd, J=2.4, 8.1, fluorenone C3-H) 7.19 (4H,dt, J=1.8,6.9 fluorenone C1,7-H) 7.40 (6H, m, fluorenone C4,5,6-H) 7.59(1H, d, J=7.2, fluorenone C8-H)

4-3: Synthesis of1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane

wherein —R— is —C₁₂H₂₄OC₁₂H₂₄OC₁₂H₂₄—.

Under a nitrogen atmosphere,1,12-bis-[12-(2′-O-9-fluorenone)-dodecyloxy]-dodecane (350 mg, 0.38mmol) was suspended in THF (3.5 ml) and dissolved at 50° C.4-Chlorophenylmagnesium bromide solution (5.65 ml, 5.65 mmol) was addeddropwise thereto, and the mixture was stirred for 2 hours. Aftercompletion of the reaction, the reaction mixture was cooled to roomtemperature, and 1N hydrochloric acid was added dropwise thereto in awater bath until foaming stopped to quench the reaction. The mixture wasextracted with chloroform (15 ml), and washed 3 times with 1Nhydrochloric acid (5 ml), 3 times with 5% NaHCO₃ solution (5 ml) andonce with 20% NaCl solution (5 ml). The organic layer was dried overNa₂SO₄, and the solvent of the filtrate was evaporated. The obtainedresidue was purified by silica gel column chromatography to give1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane(214 mg, 0.19 mmol, 49%). δ=1.2-1.6 (56H, br, Alkyl-H) 1.73 (4H, q,fl-OCH₂CH₂ —) 2.54 (2H, s, —OH) 3.36 (8H, t, —CH₂ OCH₂—) 3.90 (4H, t,fl-OCH₂—) 6.82 (2H, s, fl C1-H) 6.89 (2H, d, fl C3-H) 7.15-7.45 (14H, m,fl C6,7,8-H, Ph C2,3,5,6-H,) 7.54 (4H, d, fl C4,5-H)

4-4: Synthesis of1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane

wherein —R— is —C₁₂H₂₄OC₁₂H₂₄OC₁₂H₂₄—.

1,12-bis-[12-(2′-O-9-(4-Chlorophenyl)-9-fluorenol)-dodecyloxy]-dodecane(214 mg, 0.19 mmol) was dissolved in chloroform (2 ml), acetyl bromide(69 μl, 0.93 mmol) was added dropwise thereto, and the mixture wasstirred for 3 hours. After completion of the reaction, the solvent wasevaporated, and the residue was decanted with acetonitrile (2 ml) togive1,12-bis-[12-(2′-O-9-(4-chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane(200 mg, 0.16 mmol, 84%).

δ=1.2-1.6 (56H, br, Alkyl-H) 1.73 (4H, Br, fl-OCH₂CH₂ —) 3.38 (8H, t,—CH₂ OCH₂ —) 3.88 (4H, m, fl-OCH₂ —) 6.92 (2H, d, fl C3-H) 6.98 (2H, s,fl C1-H) 7.19-7.27 (6H, m, fl C7-H, Ph C2,6-H,) 7.34 (2H, t, fl C6-H)7.43 (2H, d, fl C8-H) 7.48 (4H, d, Ph C3,5-H) 7.55 (4H, d, fl C4,5-H)

4-5: Synthesis of Z-alanine-fluorene Anchor Compound

wherein —R— is —C₁₂H₂₄OC₁₂H₂₄OC₁₂H₂₄—.

1,12-bis-[12-(2′-O-9-(4-Chlorophenyl)-9-bromofluorene)-dodecyloxy]-dodecane(200 mg, 0.16 mmol) was dissolved in chloroform (2 ml), Z-alanine (210mg, 0.94 mmol) was added and N-ethyldiisopropylamine (216 μl, 1.25 mmol)was added, and the mixture was stirred at 50° C. overnight. Aftercompletion of the reaction, the solvent was dissolved in chloroform (4ml), and the mixture was washed once with pure water (2 ml), 5 timeswith 10% aqueous sodium carbonate solution (2 ml), twice with pure water(2 ml) and once with 20% aqueous sodium chloride solution (2 ml), driedover sodium sulfate, and the solvent was evaporated to giveZ-alanine-fluorene anchor compound (221 mg, 0.14 mmol, 90%).

δ=1.2-1.6 (59H, br, Ala CH₃ , Alkyl-H) 1.76 (4H, q, fl-OCH₂CH₂ —) 3.38(8H, t, —CH₂ OCH₂ —) 3.94 (4H, m, fl-OCH₂ —) 4.50 (2H, Br, Ala α-H) 5.07(4H, s, Z Ph-CH₂ O—) 5.16 (2H, Br, NH) 6.77 (2H, d, fl C3-H) 6.88 (2H,d, fl C1-H) 7.15-7.45 (16H, m, fl C6,7,8-H, Z Ph-H) 7.59 (4H, d, flC4,5-H)

Example 5 5-1: Synthesis of trioctadecanoxypentaerythritol

To pentaerythritol (1.5 g, 11.0 mmol) were added DMF (100 ml),1-bromooctadecane (11.4 g, 34.2 mmol) and NaH (60 wt %, 1.54 g, 38.5mmol), and the mixture was stirred at 100° C. for 22 hours. The reactionmixture was cooled to room temperature, chloroform (150 ml) was added,1N hydrochloric acid (150 ml) was further added dropwise thereto. Afterstirring for a while, the aqueous layer was discarded, and the organiclayer was further washed with 1N hydrochloric acid (100 ml) and water(100 ml). The organic layer was evaporated under reduced pressure, theresidue was precipitated with methanol (150 ml), and the obtainedcrystals were slurry washed with methanol (150 ml). Crude crystals weredried and purified by silica gel column chromatography(hexane:chloroform=1:1→hexane:ethyl acetate=10:1) to givetrioctadecanoxypentaerythritol (2.21 g, 2.47 mmol, yield 23%).

¹H-NMR (300 MHz)

0.88 (3H, t, J=6.9, —OC₁₇H₃₄-Me) 1.10-1.65 (96H, br, C18Alkyl-H) 3.12(1H, t, J=6.0, OH) 3.38 (6H, t, J=6.3, —C—(CH₂—O—CH₂ —C₁₇H₃₅)₃) 3.43(6H, s, —C—(CH₂ O—C₁₈H₃₇)₃) 3.70 (2H, d, J=5,7, HO—CH₂ —)

5-2: Synthesis of1-(3-iodo-2,2-bis-octadecanoxymethyl-propoxy)octadecane

Trioctadecanoxypentaerythritol (500 mg, 560 μmol) prepared in theabove-mentioned 5-1 was dissolved in toluene (10 ml), triphenylphosphine(294 mg, 1.12 mmol), imidazole (76.2 mg, 1.12 mmol) and iodine (284 mg,1.12 mmol) were added, and the mixture was stirred at 100° C. overnight.The reaction mixture was cooled to room temperature, toluene (10 ml) wasadded, and the mixture was washed with water (5 ml×3). The organic layerwas separated and evaporated under reduced pressure, and the residue wasprecipitated with acetonitrile (10 ml) to give1-(3-iodo-2,2-bis-octadecanoxymethyl-propoxy)octadecane (555 mg, 553μmol, yield 98%).

¹H-NMR (300 MHz)

0.88 (3H, t, J=6.9, —OC₁₇H₃₄-Me) 1.10-1.65 (96H, br, C18Alkyl-H) 3.33(6H, s, —C—(CH₂ —O—C₁₈H₃₇)₃) 3.38 (6H, t, J=6.3, —C—(CH₂—O—CH₂ C₁₇H₃₅)₃)3.48 (2H, s, I—CH₂ —)

5-3: Synthesis of2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-fluorenone

1-(3-Iodo-2,2-bis-octadecanoxymethyl-propoxy)octadecane (500 mg, 102μmol) and 2-hydroxy-9-fluorenone (30 mg, 152 μmol) were dissolved in DMF(2 ml), potassium carbonate (21 mg, 152 μmol) was added, and the mixturewas stirred at 130° C. After completion of the reaction, 0.5Nhydrochloric acid (6 ml) was added, the precipitate was filtered, andthe precipitate was washed with acetonitrile and water to give2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-fluorenone (101mg, 94 μmol).

5-4: Synthesis of2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol

Under a nitrogen atmosphere,2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-fluorenone (75mg, 73 μmol) was suspended in THF (1 ml) to allow dissolution at 50° C.4-Chlorophenylmagnesium bromide solution (0.15 ml, 0.15 mmol) was addeddropwise, and the mixture was stirred for 30 minutes. After completionof the reaction, the reaction mixture was cooled to room temperature,extracted with chloroform (3 ml), and washed 3 times with 1Nhydrochloric acid (1 ml), 3 times with 5% NaHCO₃ solution (1 ml) andonce with 20% NaCl solution (1 ml). The organic layer was dried overNa₂SO₄, the solvent of the filtrate was evaporated, and the obtainedresidue was separated and purified by silica gel column chromatographyand crystallized from methanol (1 ml) to give2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol(66 mg, 56μ mmol, 77%).

δ=0.88 (9H, t, J=6.9 Hz, OC₁₈H₃₇ C18-H) 1.1-1.6 (96H, br, OC₁₈H₃₇C2-17-H) 2.36 (1H, s, —OH) 3.35 (6H, t, OC₁₈H₃₇ C1-H) 3.45 (6H, s, —CH₂—OC₁₈H₃₇) 3.92 (2H, m, fl-OCH₂ —) 6.84 (1H, s, fl C1-H) 6.91 (1H, d, flC3-H) 7.13-7.35 (7H, m, Ph C2,3,5,6-H, fl C6,7,8-H) 7.54 (2H, m, flC4,5-H)

5-5: Synthesis of2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene

2-(3-Octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-fluorenol(66 mg, 56 μmol) was dissolved in chloroform (1 ml), acetyl bromide (41μl, 0.55 mmol) was added dropwise thereto and the mixture was stirredfor 2 hours. After completion of the reaction, the solvent wasevaporated, and the residue was crystallized from acetonitrile (1 ml) togive2-(3-octadecyloxy-2,2-bis-octadecyloxymethyl-propoxy)-9-(4-chlorophenyl)-9-bromofluorene(55 mg, 44 μmol, 79%). δ=0.88 (9H, t, J=6.9 Hz, OC₁₈H₃₇ C18-H) 1.1-1.6(96H, br, OC₁₈H₃₇ C2-17-H) 3.37 (6H, t, OC₁₈H₃₇ C1-H) 3.47 (6H, s, —CH₂—OC₁₈H₃₇) 3.97 (2H, m, fl-OCH₂ —) 6.93 (1H, d, fl C3-H) 6.95 (1H, s, flC1-H) 7.13-7.57 (9H, m, Ph C2,3,5,6-H, fl C4,5,6,7,8-H)

Example 6 Introduction of Z-Ala into Fluorene Anchor Compound

2-Docosyloxy-9-(4-chlorophenyl)-9-bromofluorene (200 mg, 0.29 mmol)obtained in Example 1 was dissolved in chloroform (2 ml), Z-alanine (197mg, 0.88 mmol) was added and N-ethyldiisopropylamine (202 μl, 1.17 mmol)was added, and the mixture was stirred at 50° C. overnight. Aftercompletion of the reaction, the solvent was evaporated, and the residuewas precipitated with acetonitrile (2 ml) to give Z-alanine-fluoreneanchor compound (178 mg, 0.22 mmol, 74%).

¹H-NMR(400 MHz)

0.88 (3H, t, J=7.0, C₂₁H₄₂-Me) 1.20-1.40 (39H, br, alkyl-H, Alanine Me)1.45 (2H, br, —O—C₂H₄—CH₂ —C₁₉H₃₉) 1.73 (2H, br, —O—CH₂—CH₂ —) 3.89 (2H,—O—CH₂ —) 4.51 (1H, br, Z—NH—CH) 5.07 (2H, s, benzyl-H) 5.19 (1H, br, s,Z—NH—) 6.78 (1H, d, J=13.5, fluorene-H) 6.90 (1H, d, J=6.3, fluorene-H)7.14-7.37 (12H) 7.57-7.60 (2H)

Example 7 Introduction of Fmoc-Ala-OH into Fluorene Anchor Compound

The fluorene anchor prepared in Example 3;2-(12-docosyloxy-dodecanoxy)-9-(3-fluorophenyl)-9-bromofluorene (600 mg,0.71 mmol) was dissolved in chloroform (6 ml), Fmoc-Ala-OH.H₂O (699 mg,2.12 mmol) and diisopropylethylamine (366 μl, 2.12 mmol) were added, andthe mixture was stirred at 70° C. overnight. After completion of thereaction, the solvent was evaporated, the residue was precipitated withacetonitrile (6 ml) to give Fmoc-Ala-O-fl (726 mg, 0.67 mmol, 95%).

¹H-NMR (300 MHz)

0.88 (3H, t, J=6.9, —OC₁₇H₃₄-Me) 1.20-1.65 (63H, br, Alkyl-H, Ala Me)1.71 (2H, br, fl-O—CH₂—CH₂ —) 3.38 (4H, t, J=6.6, —CH₂ —O—CH₂ —C₂₁H₄₃)3.89 (2H, br, fl-O—CH₂ —) 4.16 (1H, t, J=6.9, fluorene (Fmoc) C9-H) 4.34(2H, d, J=6.3, fluorene (Fmoc)-CH₂ —O) 4.53 (1H, br, Ala α-H) 5.25 (1H,br, Fmoc-NH) 6.80 (1H, br, m, fluorene-H or Ph-H) 6.85-7.05 (4H, br, m,fluorene-H or Ph-H) 7.10-7.30 (2H, br, m, fluorene-H or Ph-H) 7.37 (3H,t, J=7.2, fluorene-H or Ph-H) 7.52 (2H, br, d, J=6.9, fluorene-H orPh-H) 7.60 (2H, br, fluorene-H or Ph-H) 7.73 (2H, d, J=7.2, fluorene-Hor Ph-H)

MS

784 [M-(Fmoc-Ala)]

Example 8 Removal of Fmoc from Fmoc-Ala-O-fl

Fmoc-Ala-O-fl→H-Ala-O-fl

Fmoc-Ala-O-fl (700 mg, 0.65 mmol) prepared in Example 7 was dissolved inchloroform-acetonitrile (1:1, 7 ml), diethylamine (1.36 ml, 13.0 mmol)was added dropwise thereto at 0° C., and the mixture was stirred at roomtemperature for 2 hours. Diethylamine (1.36 ml, 13.0 mmol) was furtheradded dropwise thereto, and the mixture was stirred for 1 hour. Aftercompletion of the reaction, the solvent was evaporated, and the residuewas precipitated with acetonitrile (6 ml) to give H-Ala-O-fl (542 mg,0.63 mmol, 97% (total yield 93% vs fl-Br)).

¹H-NMR (300 MHz)

0.88 (3H, t, J=6.9, —OC₁₇H₃₄-Me) 1.20-1.65 (63H, br, Alkyl-H, Ala Me)1.74 (2H, br, fl-O-CH₂—CH₂ —) 3.38 (4H, t, J=6.6, —CH₂ —O—CH₂ —C₂₁H₄₃)3.63 (1H, br, Ala α-H) 3.90 (2H, d, J=6.6, fluorene-O—CH₂ —) 6.80 (1H,d, J=2.1, fluorene-H or Ph-H) 6.85-7.07 (4H, m, fluorene-H or Ph-H)7.10-7.30 (3H, m, fluorene-H or Ph-H) 7.35 (1H, t, J=7.4, fluorene-H orPh-H) 7.60 (2H, m, fluorene-H or Ph-H)

MS

784 [M-(H-Ala-O)]

Example 9 Condensation of Fmoc-Pro-OH

H-Ala-O-fl→Fmoc-Pro-Ala-O-fl

H-Ala-O-fl (530 mg, 0.62 mmol) prepared in Example 8 was dissolved inchloroform (7 ml), HOBt (18 mg, 0.13 mmol) and Fmoc-Pro-OH (230 mg, 0.68mmol) were added, EDC.HCl (144 mg, 0.75 mmol) was added at 0° C., andthe mixture was stirred at room temperature overnight. After completionof the reaction, the solvent was evaporated, and the residue wasprecipitated with methanol (7 ml) to give Fmoc-Pro-Ala-O-fl (695 mg,0.59 mmol, 95% (total yield 89% vs fl-Br)).

¹H-NMR (300 MHz)

0.88 (3H, t, J=6.9, —OC₁₇H₃₄-Me) 1.20-2.00 (67H, br, Alkyl-H, ProN—CH₂—CH₂ —CH₂ —CH, Ala Me) 3.38 (4H, t, J=6.6, —CH₂ —O—CH₂ —C₂₁H₄₃)3.48 (2H, br, Fmoc-N—CH₂ —) 3.88 (2H, br, fl-O—CH₂ —) 4.10-4.50 (4H, br,fluorene (Fmoc) C9-H, fluorene (Fmoc)-CH₂ —O, Ala α-H) 4.68 (1H, br, Proα-H) 6.70-7.60 (16H, br, m, fluorene-H or Ph-H) 7.75 (2H, d, J=7.2,fluorene-H or Ph-H)

Example 10 Removal of Fmoc from Fmoc-Pro-Ala-O-fl

Fmoc-Pro-Ala-O-fl→H-Pro-Ala-O-fl

Fmoc-Pro-Ala-O-fl (660 mg, 0.56 mmol) prepared in Example 9 wasdissolved in chloroform-acetonitrile (7:6, 6.5 ml), diethylamine (1.16ml, 11.1 mmol) was added dropwise thereto at 0° C., and the mixture wasstirred at room temperature for 2 hours. After completion of thereaction, the solvent was evaporated, and the residue was precipitatedwith acetonitrile (7 ml) to give H-Pro-Ala-O-fl (528 mg, 0.55 mmol, 98%(total yield 88% vs fl-Br)).

¹H-NMR (300 MHz)

0.88 (3H, t, J=6.9, —OC₁₇H₃₄-Me) 1.15-1.80 (67H, br, Alkyl-H, ProHN—CH₂—CH₂ —CH₂—CH, Ala Me) 1.87 (1H, m, Pro HN—CH₂—CH₂—CH₂ —CH) 2.11(1H, m, Pro HN—CH₂—CH₂—CH₂ —CH) 2.88 (2H, m, Pro HN—CH₂ ) 3.38 (4H, t,J=6.6, —CH₂ —O—CH₂ —C₂₁H₄₃) 3.69 (1H, m, Pro α-H) 3.90 (2H, d, J=6.6,fluorene-O—CH₂ —) 4.72 (1H, m, Ala α-H) 6.79 (1H, m, fluorene-H or Ph-H)6.85-6.95 (2H, m, fluorene-Hor Ph-H) 7.01 (2H, d, J=8.1, fluorene-H orPh-H) 7.10-7.30 (2H, m, fluorene-H or Ph-H) 7.34 (1H, m, fluorene-H orPh-H) 7.59 (2H, m, fluorene-H or Ph-H) 7.96 (1H, br, d, J=7.2,fluorene-H or Ph-H)

Example 11 Condensation of Fmoc-Ile-OH

H-Pro-Ala-O-fl→Fmoc-Ile-Pro-Ala-O-fl

H-Pro-Ala-O-fl (510 mg, 0.54 mmol) prepared in Example 10 was dissolvedin chloroform (7 ml), HOBt (15 mg, 0.11 mmol) and Fmoc-Ile-OH (206 mg,0.58 mmol) were added, EDC.HCl (123 mg, 0.64 mmol) was added at 0° C.,and the mixture was stirred at room temperature overnight. Aftercompletion of the reaction, the solvent was evaporated, and the residuewas precipitated with methanol (7 ml) to give Fmoc-Ile-Pro-Ala-O-fl (642mg, 0.50 mmol, 93% (total yield 82% vs fl-Br)).

¹H-NMR (300 MHz)

0.88 (3H, t, J=6.9, —OC₁₇H₃₄-Me) 1.20-2.20 (75H, br, m, Alkyl-H, IleMe-CH₂ —CH-Me, Pro N—CH₂CH₂ —CH₂ —CH, Ala Me) 2.30 (1H, br, s, IleMe-CH₂—CH-Me) 3.38 (4H, t, J=6.6, —CH₂ —O—CH₂ —C₂₁H₄₃) 3.58 (1H, br, ProCO—N—CH₂ —) 3.70 (1H, br, Pro CO—N—CH₂ —) 3.88 (2H, br, t, J=6.6,fl-O—CH₂ —) 4.19 (1H, t, J=6.6, fluorene (Fmoc) C9-H) 4.25-4.45 (3H, m,fluorene (Fmoc)-CH₂ —O, Ile or Pro or Ala α-H) 4.50 (1H, br, m, Ile orPro or Ala α-H) 4.66 (1H, br, m, Ile or Pro or Ala α-H) 5.40 (1H, d,J=9.3, Fmoc-NH—) 6.75-7.45 (12H, m, fluorene-H or Ph-H) 7.57 (4H, br,fluorene-H or Ph-H) 7.75 (2H, d, J=7.8, fluorene-H or Ph-H)

Example 12 Removal of Fmoc from Fmoc-Ile-Pro-Ala-O-fl

Fmoc-Ile-Pro-Ala-O-fl→H-Ile-Pro-Ala-O-fl

Fmoc-Ile-Pro-Ala-O-fl (620 mg, 0.48 mmol) prepared in Example 11 wasdissolved in chloroform-acetonitrile (7:6, 6.5 ml), diethylamine (1.00ml, 9.57 mmol) was added dropwise thereto at 0° C., and the mixture wasstirred at room temperature for 1 hour. Diethylamine (1.00 ml, 9.57mmol) was added dropwise thereto, and the mixture was stirred for 2hours. After completion of the reaction, the solvent was evaporated, andthe residue was precipitated with acetonitrile (6 ml) to giveH-Ile-Pro-Ala-O-fl (500 mg, 0.47 mmol, 98% (total yield 80% vs fl-Br)).

¹H-NMR (300 MHz)

0.90 (9H, m, —OC₁₇H₃₄-Me, Ile Me) 1.20-2.20 (75H, br, m, Alkyl-H, IleMe-CH₂ —CH-Me, Pro N—CH₂—CH₂ —CH₂ —CH, Ala Me) 2.35 (1H, br, s, IleMe-CH₂—CH-Me) 3.38 (5H, m, —CH₂ —O—CH₂ —C₂₁H₄₃, Ile α-H) 3.52 (2H, dd,J=6.0, 7.8, Pro N—CH₂ ) 3.90 (2H, t, J=6.6, fluorene-O—CH₂ —) 4.50-4.70(2H, m, Pro, Ala α-H) 6.82 (1H, m, fluorene-H or Ph-H) 6.91 (2H, m,fluorene-H or Ph-H) 7.01 (2H, m, fluorene-H or Ph-H) 7.10-7.40 (4H, m,fluorene-H or Ph-H) 7.58 (2H, m, fluorene-H or Ph-H)

Example 13 Condensation of Fmoc-Ser(tBu)-OH

(SEQ ID NO: 1) H-Ile-Pro-Ala-O-fl → Fmoc-Ser(tBu)-Ile-Pro-Ala- O-fl

H-Ile-Pro-Ala-O-fl (490 mg, 0.46 mmol) prepared in Example 12 wasdissolved in chloroform (6 ml), HOBt (13 mg, 0.10 mmol) andFmoc-Ser(tBu)-OH (194 mg, 0.51 mmol) were added, EDC-HC1 (107 mg, 0.56mmol) was added at 0° C. and the mixture was stirred at room temperatureovernight. After completion of the reaction, the solvent was evaporated,and the residue was precipitated with methanol (7 ml) to giveFmoc-Ser(tBu)-Ile-Pro-Ala-O-fl (SEQ ID NO: 1) (610 mg, 0.43 mmol, 94%(total yield 74% vs fl-Br)).

Example 14 Removal of Anchor from Fmoc-Ser(tBu)-Ile-Pro-Ala-O-fl (SEQ IDNO: 1)

Fmoc-Ser(tBu)-Ile-Pro-Ala-O-fl → Fmoc-Ser(tBu)- Ile-Pro-Ala-OH

(SEQ ID NOS: 1 and 2, respectively, in order of appearance)

Fmoc-Ser(tBu)-Ile-Pro-Ala-O-fl (SEQ ID NO: 1) (600 mg, 0.42 mmol)prepared in Example 13 was dissolved in TFA-chloroform (1%, 6 ml, TFA:about 2 eq), and the mixture was stirred at room temperature. After 30min, TFA (60 μl, about 2 eq) was added dropwise thereto, 1.5 hr later,TFA (180 μabout 6 eq) was added dropwise thereto and the mixture wasstirred for 1.5 hr. After completion of the reaction, the solvent wasevaporated, acetonitrile (6 ml) was added to the residue and theprecipitate was filtered off. The filtrate was concentrated and washedwith hexane (6 ml) to give Fmoc-Ser(tBu)-Ile-Pro-Ala-OH (SEQ ID NO: 2)(224 mg, 0.36 mmol, 86%).

MS

Fmoc-S(tBu)IPA-OH (SEQ ID NO: 2): 665 [M+H] Fmoc-S(H)IPA-OH (SEQ ID NO:2): 609 [M+H]

Similarly, Fmoc-Ser(tBu)-Ile-Pro-Ala-O-fl (SEQ ID NO: 1) (100 mg, 0.07mmol) prepared in Example 13 was dissolved in trifluoroethanol andchloroform and the mixture was stirred at 50° C. After completion of thereaction, the solvent was evaporated, methanol (3 ml) was added to theresidue and the precipitate was filtered off. The filtrate wasconcentrated, and hexane (6 ml) was added. The precipitate was collectedby filtration to give Fmoc-Ser(tBu)-Ile-Pro-Ala-OH (SEQ ID NO: 2) (30mg).

Example 15 15-1: Synthesis of(3,4,5-tris(octadecyloxy)-cyclohexyl)-methanol

Methyl trioctadecyloxy-cyclohexylcarboxylate (2.87 g, 3.03 mmol) wasdissolved in dehydrating THF (30 mL), DIBAL-H (9 mmol) was added and themixture was stirred at room temperature for 30 minutes. 1N Hydrochloricacid (10 mL) was added, and THF was evaporated under reduced pressure.Chloroform (30 mL) and 1N hydrochloric acid (30 mL) were added toseparate the layer. The organic layer was recovered and the solvent wasevaporated. The residue was crystallized from methanol and the crystalswere collected by filtration, washed well with 1N hydrochloric acid andmethanol to give (3,4,5-tris(octadecyloxy)-cyclohexyl)-methanol (2.58 g,2.81 mmol, 93%).

¹H NMR(CDCl₃)

δ=0.88 (9H, t, J=6.9 Hz, OC₁₈H₃₇ C18-H) 1.1-1.8 (101H, br, CyclohexylC1,2,6-H, OC₁₈H₃₇ C2-17-H) 3.14 (2H, m, Cyclohexyl C3,5-H) 3.35-3.57(6H, m, 3,5-OC₁₈H₃₇ C1-H, HO—CH₂ —) 3.67 (2H, t, J=6.8 Hz, 4-OC₁₈H₃₇C1-H) 3.90 (1H, s, Cyclohexyl C4-H)

15-2: Synthesis of2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenone

(3,4,5-Tris(octadecyloxy)-cyclohexyl)-methanol (82.5 mg, 89.7 μmol),2-hydroxy-9-fluorenone (26.4 mg, 135 μmol) and triphenylphosphine (35.3mg, 135 μmol) were dissolved in dehydrated THF (1.6 mL), DIED (27 μL,137 μmol) was added, and the mixture was stirred for 1.5 hours. THF wasevaporated, and 90% aqueous acetonitrile (10 mL) was added to theresidue to allow crystallization. The crystals were filtered, and theobtained crystals were washed well with acetonitrile (10 mL) to give2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenone (91.2 mg,83.1 μmol, 93%).

δ=0.88 (9H, t, J=6.6 Hz, OC₁₈H₃₇ C18-H) 1.1-1.9 (101H, br, CyclohexylC1,2,6-H, OC₁₈H₃₇ C2-17-H) 3.18 (2H, m, Cyclohexyl C3,5-H) 3.38-3.56(4H, m, 3,5-OC₁₈H₃₇ C1-H) 3.68 (2H, t, J=6.6 Hz, 4-OC₁₈H₃₇ C1-H) 3.87(2H, d, J=5.7 Hz, fl-O—CH₂ —) 3.93 (1H, s, Cyclohexyl C4-H) 6.96 (1H,dd, J=8.2, 2.4 Hz, fl C3-H) 7.20 (2H, m, fl C3,7-H) 7.36-7.46 (3H, m, flC4,5,6-H) 7.60 (1H, d, J=7.2 Hz, fl C8-H)

15-3: Synthesis of9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenol

2-(3,4,5-Tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenone (61.6 mg,56.1 μmol) was dissolved in dehydrated THF (3 mL),4-chlorophenyl-magnesium bromide (200 μmol) was added, and the mixturewas stirred at 40° C. for 3 hours. After evaporation of the solvent,0.5N hydrochloric acid (6 mL) was added to the residue to allowcrystallization. The crystals were collected by filtration, washed wellwith hydrochloric acid, water and methanol in this order to give9-(4-chloro-phenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenol(64.1 mg, 53.0 μmol, 94%).

δ=0.88 (9H, t, J=6.7 Hz, OC₁₈H₃₇ C18-H) 1.1-1.9 (101H, br, CyclohexylC1,2,6-H, OC₁₈H₃₇ C2-17-H) 2.42 (1H, s, —OH) 3.15 (2H, d, J=9.8 Hz,Cyclohexyl C3,5-H) 3.45 (4H, m, 3,5-OC₁₈H₃₇ C1-H) 3.66 (2H, t, J=6.5 Hz,4-OC₁₈H₃₇ C1-H) 3.78 (2H, d, J=5.3 Hz, fl-O—CH₂ —) 3.91 (1H, s,Cyclohexyl C4-H) 6.80 (1H, s, fl C1-H) 6.88 (1H, d, J=8.3 Hz, fl C3-H)7.13-7.38 (7H, m,Ph C2,3,5,6-H, fl C6,7,8-H) 7.55 (2H, m, fl C4,5-H)

15-4: Synthesis of9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-bromofluorene

9-(4-Chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-fluorenol(62.0 mg, 51.2 μmol) was dissolved in chloroform (2 mL), acetyl bromide(13 μmol, 172 μmol) was added, and the mixture was stirred at roomtemperature for 1 hours. After evaporation of the solvent, acetonitrilewas added to the residue to allow crystallization to give9-(4-chlorophenyl)-2-(3,4,5-tris(octadecyloxy)-cyclohexylmethoxy)-9-bromofluorene(63.9 mg, 50.2 μmol, 98%). δ=0.88 (9H, t, J=6.6 Hz, OC₁₈H₃₇ C18-H)1.1-1.9 (101H, br, Cyclohexyl C1,2,6-H, OC₁₈H₃₇ C2-17-H) 3.16 (2H, d,J=10.3 Hz, Cyclohexyl C3,5-H) 3.46 (4H, m, 3,5-OC₁₈H₃₇ C1-H) 3.68 (2H,t, J=6.7 Hz, 4-OC₁₈H₃₇ C1-H) 3.82 (2H, m, fl-O—CH₂ —) 3.92 (1H, s,Cyclohexyl C4-H) 6.90 (1H, d, J=8.3 Hz, fl C3-H) 6.96 (1H, s, fl C1-H)7.18-7.26 (3H, m,Ph C3,5-H, fl C7-H) 7.34 (1H, t, J=7.1 Hz, fl C6-H)7.43 (1H, d, J=7.6 Hz, fl C8-H) 7.48 (2H, d, J=8.7 Hz, Ph C2,6-H) 7.57(2H, m, fl C4,5-H)

Reference Example 1 Synthesis of Z-alanine-trityl anchor(3,4,5-tristearyloxy) compound

bis-(4-Chlorophenyl)-(3,4,5-tristearyloxyphenyl)-methyl chloride (150mg, 0.13 mmol) prepared according to the method described inWO2007/122847 was dissolved in chloroform (1.5 ml), Z-alanine (58 mg,0.26 mmol) was added, N-ethyldiisopropylamine (49 μl, 0.28 mmol) wasadded, and the mixture was stirred at room temperature for 1 hour. Aftercompletion of the reaction, the solvent was evaporated, and the residuewas precipitated with acetonitrile (1.5 ml) to give Z-alanine-tritylanchor compound (169 mg, 0.13 mmol, 97%).

Reference Example 2 Synthesis of Z-alanine-trityl anchor(3,5-distearyloxy) compound

bis-(4-Chlorophenyl)-(3,5-distearyloxyphenyl)-methyl chloride(Calculated 404 mg, 0.46 mmol) prepared according to the methoddescribed in WO2007/122847 was dissolved in chloroform (4 ml), Z-alanine(204 mg, 0.91 mmol) was added, N-ethyldiisopropylamine (173 μl, 1.00mmol) was added, and the mixture was stirred at room temperature for 3hours. After completion of the reaction, the solvent was evaporated, andthe residue was precipitated with acetonitrile (8 ml) to giveZ-alanine-trityl anchor compound (548 mg, 0.51 mmol, 100% 2 steps).

Experimental Example

(1) Stability Evaluation of Fluorene Type Anchor-Condensed Product andTrityl Type Anchor-Condensed Product, with Z-Alanine

A Z-alanine-anchor-condensed product (35 mg) was dissolved or suspendedin methanol and 10% acetic acid/chloroform solution (0.7 ml), and theamount of Z-alanine (Z-Ala-OH) liberated in a predetermined time wasquantified.

The trityl type anchor showed liberation of Z-alanine in methanol evenin 1 hour and the bond to the anchor was cut. On the other hand, it wasconfirmed that the fluorene type anchor was not cut even in methanol orunder acidic conditions of 10% acetic acid solution (see Table 1).

TABLE 1 Amount of liberated Z-Ala-OH after stirring at room temperaturefor 1 hr MeOH 10% AcOH/CHCl₃

70% 100%

20% 75%

2% 4%

0% 0%

INDUSTRIAL APPLICABILITY

A particular compound having a fluorene skeleton provided substancewhich functions as a superior protecting group and anchor can beproduced. The anchor can be removed under weak acidic conditions, andselectively removed even when other protecting groups (peptideside chainprotecting group etc.) still remain in the compound obtained by organicsynthesis reaction. Namely, the compound is a substance that enableseasy precipitation in methanol and the like, since it dissolves only inhalogen solvents, THF and the like and scarcely dissolves in polarorganic solvents. In the process of elongation of peptide chain lengthwherein the substance is used as a protecting group for C-terminal orside chain in the peptide synthesis and an operation including reactionin a halogen solvent, followed by precipitation with methanol and thelike to remove impurity is repeated, a side reaction to yielddiketopiperazine is suppressed and peptide chain can be elongated in ahigh yield and with high quality. Moreover, the anchor enables selectiveremoval thereof while maintaining the protecting group. Using the methodof the present invention, various active pharmaceutical ingredients(API), intermediates and final products are easily obtained.

Where a numerical limit or range is stated herein, the endpoints areincluded. Also, all values and subranges within a numerical limit orrange are specifically included as if explicitly written out.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

All patents and other references mentioned above are incorporated infull herein by this reference, the same as if set forth at length.

The invention claimed is:
 1. A method of producing a peptide by a liquidphase synthesis process, comprising: (a) binding a fluorene compound toan amino acid or a peptide, to obtain a bonded product; (b)precipitating said bonded product and; (c) deprotecting said bondedproduct after a reaction to produce a peptide; wherein said fluorenecompound is represented by formula (I):

wherein ring A is an aromatic ring; Y is a group reactive with one ormore groups selected from an amino group, a carboxyl group, and amercapto group; at least one of rings A, B, and C has an organic grouphaving an aliphatic hydrocarbon group; and rings A, B, and C eachindependently optionally have an electron withdrawing group.
 2. A methodof producing a peptide by a liquid phase synthesis process, comprising:(a) obtaining a C-fluorene compound-protected amino acid or a C-fluorenecompound-protected peptide by condensing a fluorine compound with theC-terminal of a first N-protected amino acid or a first N-protectedpeptide; (b) removing a protecting group of the N-terminal of the aminoacid or peptide obtained in the above-mentioned step to obtain anN-deprotected amino acid or peptide; (c) condensing the N-terminal ofthe N-deprotected amino acid or peptide with a second N-protected aminoacid or a second N-protected peptide, to obtain a peptide; and (d)precipitating the peptide obtained in the above-mentioned step; whereinsaid fluorene compound is represented by formula (I):

wherein ring A is an aromatic ring: Y is a group reactive with one ormore groups selected from an amino group, a carboxyl group, and amercapto group; at least one of rings A, B, and C has an organic grouphaving an aliphatic hydrocarbon group; and rings A, B, and C eachindependently optionally have an electron withdrawing group.
 3. Themethod according to claim 2, further comprising one or more repetitionsof: (e) deprotecting the N-terminal of the peptide obtained in (d)precipitating; (f) condensing the N-terminal of peptide obtained in (e)deprotecting with N-protected amino acid or N-protected peptide toobtain an elongated peptide; and (g) precipitating the elongated peptideobtained in the above-mentioned step.
 4. A method of producing a peptidefurther comprising, after the final precipitation step according toclaim 2, a step of deprotecting the C-terminal of peptide wherein theC-terminal is protected with a fluorine compound.